Toward routine, DNA-based detection methods for marine pests

This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institution

and sharing with colleagues.

Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third party

websites are prohibited.

In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further information

regarding Elsevier’s archiving and manuscript policies areencouraged to visit:

http://www.elsevier.com/copyright

http://www.elsevier.com/copyright

Author's personal copy

Research review paper

Toward routine, DNA-based detection methods for marine pests

Nathan J. Bott a,⁎, Kathy M. Ophel-Keller b, Michael T. Sierp c, Herdina b, Keith P. Rowling a, Alan C. McKay b,Maylene G.K. Loo a, Jason E. Tanner a, Marty R. Deveney a

a Aquatic Sciences, South Australian Research and Development Institute, PO Box 120, Henley Beach, South Australia 5022, Australiab Sustainable Systems, South Australian Research and Development Institute, GPO Box 397, Adelaide, South Australia 5001, Australiac Aquatic Biosecurity Program, Primary Industries and Resources South Australia, GPO Box 1625 Adelaide, South Australia 5001, Australia

a b s t r a c ta r t i c l e i n f o

Article history:Received 2 February 2010Received in revised form 6 May 2010Accepted 11 May 2010Available online 19 May 2010

Keywords:Marine pestsDNADetectionMolecular technologiesSpecific diagnosisGenetic markersPolymerase chain reaction (PCR)Hybridisation

Marine pest incursions can cause significant ongoing damage to aquaculture, biodiversity, fisheries habitat,infrastructure and social amenity. They represent a significant and ongoing economic burden. Marine pestscan be introduced by several vectors including aquaculture, aquarium trading, commercial shipping, fishing,floating debris, mining activities and recreational boating. Despite the inherent risks, there is currentlyrelatively little routine surveillance of marine pest species conducted in the majority of countries worldwide.Accurate and rapid identification of marine pest species is central to early detection and management.Traditional techniques (e.g. physical sampling and sorting), have limitations, which has motivated someprogress towards the development of molecular diagnostic tools. This review provides a brief account of thetechniques traditionally used for detection and describes developments in molecular-based methods for thedetection and surveillance of marine pest species. Recent advances provide a platform for the developmentof practical, specific, sensitive and rapid diagnosis and surveillance tools for marine pests for use in effectiveprevention and control strategies.

Crown Copyright © 2010 Published by Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7072. Traditional techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7073. A DNA approach to the identification and discrimination of pest species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7074. Molecular methods utilised for routine identification and/or diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 709

4.1. Hybridisation techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7094.1.1. In situ hybridisation (ISH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7094.1.2. Sandwich hybridisation arrays (SHA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7094.1.3. Hybridisation array . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7094.1.4. Hybridisation quartz microbalance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 710

4.2. Polymerase chain reaction (PCR)-based techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7104.2.1. Melting curve analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7104.2.2. Potential issues with PCR-based approaches. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 711

5. Extraction of genomic DNA from environmental samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7116. Future approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 711

6.1. Routine monitoring and surveillance of marine pests in port waters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7116.2. Testing ballast water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7116.3. Future directions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7116.4. Legal requirements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 712

7. Concluding remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 712Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 712References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 712

Biotechnology Advances 28 (2010) 706–714

⁎ Corresponding author. Tel.: +61 8 8207 5485; fax: +61 8 8207 5406.E-mail address: [email protected] (N.J. Bott).

0734-9750/$ – see front matter. Crown Copyright © 2010 Published by Elsevier Inc. All rights reserved.doi:10.1016/j.biotechadv.2010.05.018

Contents lists available at ScienceDirect

Biotechnology Advances

j ourna l homepage: www.e lsev ie r.com/ locate /b iotechadv


1. Introduction

Marine pests have the potential to cause significant harm to en-demic biodiversity and habitats (Galil, 2007;Wallentinus and Nyberg,2007). Marine pests can be translocated and introduced by numerousvectors including ship ballast, hull fouling, floating debris and man-made structures such as drilling platforms and canals (Bax et al.,2003). Marine pest introductions continue to occur and threaten themarine environment and associated industries (Hayes and Sliwa,2003). With increasing globalisation comes faster and more frequentshipping and air transport of live seafood. Propagule pressure is onlylikely to increase unless effective strategies are employed for earlydetection, prevention and control. Central to such strategies is theability to rapidly identify the presence of a particular pest species.

There are over 250 exotic marine species recorded in Australiaalone (Hayes and Sliwa, 2003) and in San Francisco Bay there are atleast 230 established exotic species that account for 40–100% of thecommon species at many sites in the estuary (Cohen and Carlton,1998). Even if suitable eradication technologies were available, insome cases it may be be un-economical to do so, given the logisticaldifficulties associated with working in an marine environment. Themain economic and social impacts of marine pests are decreases inthe economic output of marine-based activities from aquaculture,fisheries and tourism, and negative impacts on human health (Baxet al., 2003). The total economic cost of a marine pest incursion isdifficult to accurately define but in Australia, a major marine pesteradication has been estimated to cost up to AUD$263M modelledusing general parameters (Crombie et al., 2007, p. 20). The fewexamples of successful eradication of marine pests worldwide havebeen when the invasion has been detected in its very early stages,and/or it is restricted to an area that can be isolated from the rest ofthe marine environment (e.g. Caulerpa taxifolia eradication in South-ern California (Anderson, 2005)); Black-striped mussel, Mytilopsissallei in Darwin, Australia (Galil and Bogi)). Booth et al. (2007)reported that marine ecosystems are at high risk from marinetransport translocating invasive seaweeds and the ability to identifyspecies is of utmost importance.

Marine pests include a wide range of plant and animal taxa. Theseinclude macro- and microalgae, molluscs, crustaceans, annelids, echi-noderms, cnidarians, ascidians and teleosts. The development andimplementation of rapid, sensitive and accurate diagnostic techniquesfor the identification and surveillance of marine pests from environ-mental samples (e.g. sea water, sediments, and ship's ballast), par-ticularly in areas that are currently pest free, is an essential step in earlydetection and control of marine pests. There has been some effortconcentrated towards molecular detection of freshwater invasives,including zebra and quagga mussels (see Baldwin et al., 1996; Claxtonand Boulding, 1998; Frischer et al., 2002; Wang et al., 2006a,b). In thisarticle, we: (i) briefly describe techniques traditionally used for theidentification and/or surveillance of marine pests, (ii) review DNAtechnical approaches for the identification of species and definitionof genetic markers, (iii) describe molecular based methods for thedetection of marine pests, and (iv) propose some options for thedevelopment of improved molecular diagnostic techniques.

2. Traditional techniques

Baseline surveys and repeated monitoring conducted by experi-enced biologists are the prime requirement for early detection ofmarine pest species using standard methods. Campbell et al. (2007)reviewed the five survey methods commonly employed for thedetection of marine pest species: Hewitt and Martin protocols, RapidAssessment Surveys, Bishop Museum protocols, Chilean aquaculturesurveys and Passive Sampling protocols. These methods typically in-volve physical sampling, sorting and identification to understand theresident biota and identify marine pests. The Marine Pest Monitoring

Manual developed under Australia's National System for the Preven-tion and Management of Marine Pest Incursions illustrates a com-bination of these methods (NIMPCG, 2009a, 2009b).

Traditional survey methods typically require specialised taxonomicexperts, trained in identifying organisms across a range of life-cyclestages. The sorting and identification of plants and animals is timeconsuming, sometimes taking years before species are accuratelyidentified, but the sampling data provided by surveys of this kindprovide an important baseline understanding of biodiversity for futurepest surveillance and ecological research. Unfortunately there is a globaldecline in taxonomic expertise (Hopkins and Freckleton, 2002; Kimand Byrne, 2006). Given thewide range of taxa required to be identifiedin marine pest surveys and the inherent difficulties in identification,there is a need to develop methods that work in conjunction with, andcomplementary to, available taxonomic expertise.

3. A DNA approach to the identification and discriminationof pest species

As the use of molecular biological techniques has become morewidespread, so too has their use in the identification of marinepest species. A range of DNA-based techniques have been used (seeTable 1) to discriminate between species because of the ability ofthese methods to detect subtle genetic differences. DNA is relativelystable and can be readily isolated from fresh, frozen, ethanol-preservedor commercially available nucleic acid storage buffer (e.g. RNAlater(Ambion)) preserved specimens for subsequent analysis using cloning,hybridisation and enzymatic amplification procedures.

The polymerase chain reaction (PCR) (Mullis et al., 1986; Saikiet al., 1988) is a reliable method of enzymatic nucleic acid ampli-fication which has significantly advanced a wide range of scientificareas. The majority of molecular assays for the detection and iden-tification of marine pests and their allies involve PCR-based tech-nologies. The key to a reliable and robust molecular method for theidentification of a marine pest is the definition of one or more suitableDNA target regions (genetic marker or locus).

This review covers a range of taxonomic groupings and no singlemarker should be considered universally appropriate to all taxa.Nuclear ribosomal genes and spacers, mitochondrial genes andchloroplast genes (for plants), have been utilised to identify marinepests (see Table 1). Caution must always be taken when determiningthe DNA marker of choice for the development of a new molecularassay. A literature review of previous assays developed for closelyrelated organisms must be conducted and a thorough knowledge ofthe sequence variation within the marker of choice (both intra- andinter-specific variation) must be obtained from previously publisheddata (e.g. GenBank-http://www.ncbi.nlm.nih.gov/Genbank/). DNAsequencing of the marker of choice of target and related organismsmust be carried out in order to design an assay which is specific to thetarget taxon.

Genes evolve at different rates and a suitable DNA region shouldvary in sequence sufficiently to allow the identification of an individualto the taxonomic level required. For specific identification, the DNAmarker should exhibit little or no genetic variation within a speciesbut differ sufficiently between species so as to allow unequivocaldelineation. For the identification of population variants (strains orgenotypes), the marker should vary considerably in sequence with-in a species but still offer enough specificity so as to not cross-react(false-positives) with heterologous species.

In nuclear genes and intergenic spacers, there is typically littlevariation amongst individuals within a population and between otherpopulations (Larsen et al., 2005; Livi et al., 2006). Nuclear rDNA ofeukaryotes is repetitive and consists of hundreds of tandem repeats.These tandem repeats are often distributed on different chromosomes(Hillis and Dixon, 1991; Eickbush and Eickbush, 2007), and like otherrepetitive DNA elements show sequence homogeneity as a result of

707N.J. Bott et al. / Biotechnology Advances 28 (2010) 706–714


concerted evolution (Elder and Turner, 1995; Eickbush and Eickbush,2007). The rDNA array consists of the intergenic non-transcribedspacer (IGS or NTS), the external transcribed spacer (ETS), and thetranscription unit comprising three rRNA genes (short sub-unit (SSU),5.8S and long sub-unit (LSU)), which are separated by the first andsecond internal transcribed spacers (ITS-1 and ITS-2). The rDNA genes,ITS and IGS/NTS regions have been shown to be particularly useful indefining species specific markers for marine pest assay development(see Table 1).

Genes in the small, double-stranded, with few exceptions circularmitochondrial genome generally evolve at a quicker rate than nucleargenes. Animal cells typically contain hundreds to thousands of mito-chondria per cell (Xu et al., 2008) which increases detection potentialmaking mitochondrial markers potentially suitable for sensitive mo-lecular assays. Evolutionary rates of mitochondrial DNA may varybetween taxonomic lineages, but are generally believed to undergorelatively rapid rates of evolution (Kocher et al., 1989; Rand, 1994).Mitochondria are generally inherited maternally making them par-ticularly useful as a species-specific marker for the delineation of

closely related species (e.g. Blair et al., 2006, Kamikawa et al., 2008).The identification of different strains (i.e. population variants) hasimportant implications for understanding the geographical originand patterns of spread of marine pest species, potentially providing abetter understanding of patterns and means of translocation. Mito-chondrial DNA has proven particularly useful in understanding thegenetic structure of populations due to their maternal inheritancewhich provides tracers to patterns of colonisation and its smallereffective population size (when compared to the nuclear genome(Harrison, 1989; Buonoccorsi et al., 2001; Kawakami et al., 2007).

Like mitochondria, chloroplasts have a circular genome that issmaller than the nuclear genome. The chloroplast genome has beena major focus in studying plant evolution and genetics (Golenberget al., 1993; Clegg et al., 1994; Morton, 1995). Typically chloroplastgenomes display lower levels of heterogeneity than mitochondrialgenomes, this is in part due to mitochondria having larger genomeswhich encode for less genes than chloroplasts (Palmer, 1990; Soria-Hernanz et al., 2008). The ribulose-bisphosphate carboxylase (rbcL)gene is of interest for use as a diagnosticmarker of algae. Hanyuda et al.

Table 1Examples of molecular methods and markers used for the detection of different taxonomic groups of marine pests.

Taxonomic group DNA loci Methods References

Dinophyceae ITS 1 & 2, 5.8S rDNA PCR coupled rDNA probe Penna and Magnani, 1999qPCR Park et al., 2007

rDNA PCR Godhe et al., 2001Sandwich Hybridisation Assay (SHA) Tyrell et al., 2002; Ayers et al, 2005

LSU rDNA PCR-RFLP Scholin and Anderson, 1994PCR Patil et al., 2005aHybridisation Array Ki and Han, 2006Hybridisation (quartz microbalance) Lazerges et al., 2006FISH Touzet and Raine, 2007SHA Anderson et al., 2005

SSU rDNA FISH Takahashi et al., 2005PCR-FISH Godhe et al., 2007SHA Ahn et al., 2006; Diercks et al., 2008Hybridisation array Ahn et al., 2006qPCR Bowers et al., 2000

5.8S rDNA qPCR Galluzzi et al., 2004Macroalgae ITS 1 & 2, 5.8S rDNA PCR coupled dot-blot hybridisation Blomster et al., 2000

IGS PCR Provan et al., 2005; Harvey et al, 2009rbcL gene PCR Fama et al., 2002; Freshwater et al., 2006Mitochondrial: cox1, 16S PCR Provan et al., 2005Mitochondrial: Cytochrome b and cox1 PCR Kamikawa et al., 2008

Gastropods Mitochondrial: cox1 PCR Gunasekera et al., 2005Bivalves Mitochondrial: cox1, nad4, 16S Multiplex PCR Hare et al., 2000;

Nested PCR Patil et al., 2005b;PCR Stepien et al., 1999; Blair et al., 2006;

Pie et al., 2006; Boeger et al., 2007Microsatellite loci PCR Morgan and Rogers, 2001SSU rDNA Nested Multiplex Larsen et al., 2005, 2007

PCR-SSCP Livi et al., 2006FISH Le Goff-Vitry et al., 2007; Pradillon et al., 2007SHA Jones et al., 2008

5S rDNA, NTS PCR Cross et al., 20065S rDNA PCR-RFLP Fernandez-Tajes and Medez, 2007IGS PCR Harvey et al. 2009

Crustaceans Mitochondrial: cox1 qPCR Pan et al., 2008PCR Chu et al., 2003; Tang et al., 2003PCR-RFLP Yamasaki et al., 2006

ITS PCR Tang et al., 2003SHA Goffredi et al., 2006; Jones et al, 2008

IGS PCR Harvey et al., 2009Not Applicable RAPD-PCR Zhou and Gao, 1999

Echinoderms Mitochondrial: cox1 PCR Deagle et al., 2003SSU rDNA FISH Mountfort et al., 2007

Polychaetes SSU rDNA FISH Pradillon et al., 2007SHA Jones et al., 2008

708 N.J. Bott et al. / Biotechnology Advances 28 (2010) 706–714


(2000) reported that the intron in the rbcL gene was highly variableamongst different species and strains of Caluerpa and Famà et al.(2002) used a rbcL PCR-based assay to identify invasive strains ofCaulerpa taxifolia. Freshwater et al., 2006 also used a rbcL gene PCR-based assay to discriminate invasive Gracilaria from nearly morpho-logically identical native species.

4. Molecular methods utilised for routine identificationand/or diagnosis

A wide range of molecular-based techniques have been employedfor the identification of species at adult and/or larval stages of marinepest or related species (see Table 1). The advantages, limitations anduses of these techniques for marine pest research and detection aresummarised in Table 2 and the limitations of these approaches arefurther discussed in the following section.

4.1. Hybridisation techniques

4.1.1. In situ hybridisation (ISH)In situ hybridisation (ISH) involves the use of a labelled comple-

mentary DNA or RNA strand (i.e. probe) that localises to a specificDNA or RNA sequence. ISH incorporates a number of different meth-ods which allow visualisation within a tissue (section), or for the caseof a larval or spore stage, the entire organism (eggs, larvae etc.)(whole mount ISH). Fluorescence in situ hybridisation (FISH) involvesthe use of fluorescently labelled probes and is often utilised withfluorescence microscopy. ISH techniques have been employed for theidentification of marine pest species, including toxic dinoflagellates(Anderson et al., 2005; Frischer et al., 2002; Godhe et al., 2007;

Takahashi et al., 2005; Touzet and Raine, 2007) and bivalves (Le Goff-Vitry et al., 2007; Pradillon et al., 2007).

4.1.2. Sandwich hybridisation arrays (SHA)SHA uses an oligonucleotide probe with a specific capture probe

targeted at the DNA/RNA target and one or more signal probes whichattach to the bound region (thus forming a sandwich). The presenceof target DNA/RNA in the sample is represented colorimetrically,fluorescently or chemiluminscently based on a number of differentsignal probe chemistries. SHA has potential for on site testing (seeGoffredi et al., 2006). There have been numerous studies documentingthe development of SHA for the detection of the toxic microalgae (seeAhn et al., 2006; Ayers et al., 2005; Diercks et al., 2008; Tyrell et al.,2002) as well as for various other marine pest species (Goffredi et al.,2006; Jones et al, 2007).

4.1.3. Hybridisation arrayHybridisation arrays employ a DNA hybridisation method where-

by an ordered arrangement of multiple short synthetic DNA strandsare attached onto a solid surface to construct an array of DNA probes.DNA samples are passed across the array and target DNA moleculeshybridise to their specific probe which releases a label that is detectedand quantified. A microarray is a miniaturised set of thousands ofhybridisation arrays on a single “chip” allowing the screening formany different species or strain variants. Based on the position andamount of label, DNA microarrays can be used to determine whichprobe has hybridised to the target DNA. Array technology has theadvantage of being able to test for numerous targets in one sampleand the cost per target tested is potentially lower than other tech-nologies, particularly when thousands of markers are utilised on theone array. Ki and Han (2006) utilised hybridisation array technology

Table 2Molecular diagnostic techniques suitable for different sample types for the identification and detection of marine pest species.

Technique Suitable sample types Advantages Limitations Example references

In situ hybridisation Whole animals, larval andegg/spore stages

Highly specific, provides visual verification

Long processing time; difficultand expensive to develophigh-throughput applications

Mountfort et al., 2007;Pradillon et al., 2007

Sandwich arrays Whole animals, larval andegg/spore stages

Highly specific, providesvisual verification

Not suited to high-throughputapplications

Ayers et al., 2005; Dierckset al, 2008; Goffredi et al, 2006;Jones et al., 2008; Tyrell et al., 2002

Quartz crystalmicrobalance

Whole animals, larval andegg/spore stages,environmental samples

Highly specific, potential foruse as real-time biosensors

Not suited to widespreadsurveillance of multiple species.

Lazerges et al., 2006

Hybridisation arrays Whole animals, larval and egg/spore stages, environmentalsamples

Highly specific, differentformats can offer the ability todetect many different strainsand species

Can be expensive to develop,statistical analyses complex.

Ki and Han. 2006

PCR (end-point one step,nested and multiplex,agarose gel electrophoresisand/or sequencing)

Whole animals, larval andegg/spore stages,environmental samples

Can be highly specific, relativelyinexpensive, able to amplifyminute amounts of DNA.

Post-PCR handling time consuming,potential exposure to toxic reagents.Nested PCR has potential for PCRcontamination. Multiplex PCR can bedifficult to develop effectively.

Hare et al., 2000; Deagleet al., 2003; Boeger et al., 2007

PCR-Dot Blot Hybridisation Whole animals, larval andegg/spore stages,environmental samples

Can be highly specific,inexpensive, amplify minuteamount of DNA, obtainresults quickly.

Post-PCR handling time, ifhybridisation step requires specificity.Slower than QPCR applications.

Blomster et al., 2000

PCR-RAPD Whole animals, larval andegg/spore stages

Inexpensive Not suited to specific amplification,requires post-PCR analysis

Zhou and Gao, 1999

PCR-RFLP Whole animals, larval andegg/spore stages

Inexpensive, can be useful indiscriminating closely relatedspecies/strains

Not suited to specific amplification,requires post-PCR analysis

Yamasaki et al., 2006;Fernandez-Tajes and Medez, 2007

PCR-SSCP Whole animals, larval andegg/spore stages

Cost-effective, a powerfulmethod of distinguishingclosely related species/strains

SSCP electrophoresis is timeconsuming, not suited to widespreadtesting on environmental samples

Livi et al., 2006

Quantitative Real-time PCR Whole animals, larval andegg/spore stages,environmental samples

Highly specific, rapid analysis,allows quantification ofamount of target species in asample, and potential forhigh-throughput application

Assay development can be expensiveand time consuming. Incorrectlydeveloped assays may producefalse-positives.

Pan et al., 2008; Park et al., 2007



for the detection of several species responsible for harmful algalblooms (HAB), whilst Ahn et al, 2006 developed a fibre-optic basedarray for the detection of HAB.

4.1.4. Hybridisation quartz microbalanceQuartz microbalances can be utilised for the detection of DNA

through the immobilisation on a gold surface of Digoxigenin (DIG)labelled-DNA probes complimentary to specific DNA-strands whichare attached to a quartz crystal. Detection of specific DNA is recordedby a change in frequency of the quartz resonator. Lazerges et al.(2006) developed a quartz crystal microbalance sensor for the detec-tion of Alexandrium minutum blooms.

4.2. Polymerase chain reaction (PCR)-based techniques

The polymerase chain reaction (PCR) has revolutionisedmany areasof biological research including species and strain delineation. PCRcan amplify minute amounts of template DNA, and its high specificitymakes it the highly effective for species and strain identification for awide range of organisms. PCR with direct sequencing is a gold standardtechnique; however, it takes several days to obtain results, a relevantquestion is whether other procedures can be used as reliably in thesameamount of time. The relatively low cost of equipment and reagentsmakes PCR accessible to small laboratories.

Some of the PCR-based methods used for the identification ordiscrimination of marine pest species are:

Species detection through design of specific primersPCR utilising genus or species specific primers for marine pestshas been utilised using a wide range of techniques: one-stepreactions (e.g. Patil et al., 2005a; Harvey et al., 2009; Kamikawa etal., 2008; Cross et al., 2006; Chu et al., 2003; Deagle et al., 2003);nested PCR (Patil et al., 2005b); Multiplex (Hare et al., 2000); andnested multiplex (Larsen et al., 2005, 2007).

Random Amplified Polymorphic DNA (RAPD)RAPD is based on the amplification of random fragments ofgenomic DNA using single primers of arbitrary sequence. RAPDoffers rapid molecular assessment in that it does not require thedesign of specific primers that bind exclusively to one sequencetype. It essentially scans the entire genome without priorknowledge of sequence information. RAPDwill produce a specificbanding pattern when amplicons are subjected to electrophore-sis. It can be particularly useful in discriminating between closelyrelated species or strains and understanding sequence variationbetween individuals (Mbwana et al., 2006). Zhou and Gao, (1999)utilised RAPD for the identification of mitten crab (Eriocheirsinensis) populations but it has not otherwise been widelyutilised for marine pest identification.

Restriction Fragment Length Polymorphism (RFLP)Restriction endonucleases are used to cut DNA at precise sequencesproducing specific bands which are visualised via electrophoresis.When RFLP is combined with PCR, a fragment will be amplified viaPCR before being digested using the restriction endonucleases togive a characteristic pattern based on the amplified fragment. RFLPhas not been widely used for the identification and/or surveillanceof marine pests. Scholin and Anderson, (1996) used PCR-RFLPto distinguish between six species of the dinoflagellate genus,Alexandrium. Fernandez-Tajes andMedez, (2007)utilisedPCR-RFLPof 5S rDNA for the discrimination of razor clams (Ensis spp.), andYamasaki et al., (2006) utilised RFLP of the mitochondrial genecytochrome oxidase subunit 1 (cox1) to identify Japanese MittenCrab (Eriocheir japonica).

Single Strand Conformation Polymorphism (SSCP)SSCP is a mutation scanning approach that analyses amplicons of100–450 nucleotides by denaturing double stranded DNA intosingle strand DNA and electrophoretically analysing the single

stranded conformational profiles (conformers) in a non-dena-turing gel matrix. SSCP has been shown to distinguish betweensequences that differ by a single base (Gasser et al., 2006). It canbe a highly effective cost saving tool. Once sequences (i.e. speciesor strains) are known for conformational profiles the extra costof direct DNA sequencing is eliminated. SSCP generally doesnot require optimisation for different sequences or amplicons(Gasser et al., 2006). Livi et al. (2006) utilised PCR coupled SSCPof the short sub-unit (SSU)-rDNA for the discrimination of bivalvelarvae.

PCR coupled dotlot hybridisationPCR coupled dot-blot hybridisation involves firstly PCR amplifi-cation and secondly detection of that amplicon using staining orradioassay (e.g. 32P, DIG) of complementary target bound to asupport. The method is useful for differentiating between closelyrelated species when species-specific probes are designed (e.g.Blomster et al., 2000) and is a quick and relatively inexpensivemeans of identification and discrimination. Blomster et al. (2000)used an ITS-based dot blot hybridisation to distinguish betweenspecies of the Chlorophyte genus, Enteromorpha, while Pennaand Magnani (1999) employed a 32P-labelled probe to identifyAlexandrium spp.

Quantitative Real-ime PCR (QPCR)Quantitative real-time PCR (QPCR) allows the amplification of atarget PCR amplicon to bemonitored in real-time as amplificationoccurs. QPCR utilises two main detection systems: fluorescentintercalating dyes and probes. There are a number of intercalat-ing dyes utilised for QPCR. The original method used ethidiumbromide and measured the change in fluorescence after eachcycle using a digital camera and a fluorometer (Higuchi et al.,1993). Subsequent modifications incorporated non-carcinogenicdyes such as SYBR Green I (Becker et al., 1996), LCGreen (Wittweret al., 2003), SYTO9 (Monis et al., 2005) and EvaGreen (Wanget al., 2006a,b).

Probe-based detection systems include TaqMan probes (Heid et al.,1996), molecular beacons (Piatek et al., 1998), fluorescence resonanceenergy transfer (FRET) (Chen and Kwok, 1999) and minor groovebinder (MGB) eclipse probes (Afonina et al., 2002). These probes aremore expensive than intercalating dyes but ensure specificity ofthe assay through ensuring exclusive binding to the target sequence(Monis et al., 2005).

QPCR is typically analysed in two ways. Relative quantificationwill provide a cycle threshold (Ct) for the specific amplification ofthe target sequence based on comparison with amplification of a‘normaliser’ gene from the same DNA template. It should be presentwith the same copy number of the locus of interest. Absolute quan-tification is performed by determining the Ct of test samples byamplifying target DNA standards (of known concentrations) at thesame time as test samples, the Ct is calculated based on a standardcurve generated from the DNA standards.

Real-time PCR offers a relatively rapid analysis (b2 h), the po-tential for high-throughput applications, allows linear quantificationover a wide dynamic range (N6 orders of magnitude) and the benefitof not requiring post-PCR handling (“closed-tube” format). It is nowroutinely used in a wide-range of clinical applications for the detec-tion of a wide range of bacterial, fungal, parasitic and viral diseasesof humans (Espy et al., 2006). Recent advances have seen a numberof studies utilising QPCR-based techniques for the identification ofmarine pests (see Galluzzi et al., 2004; Pan et al., 2008).

4.2.1. Melting curve analysesWhen using intercalating dyes some real-time PCR thermocyclers

can also perform melting analysis or High Resolution Melting (HRM)analysis mutation scanning techniques. Melting analyses allows rapidpost-PCR analysis of sequence variants or strains/species (if required)



or a secondary confirmation of the presence of the target amplicon inthe sample based on consistency of the melt curve with the definedDNA standards. This approach has been employed for a number ofstudies concerned with human and livestock pathogens (see Bottet al., 2009; Pangasa et al., 2009). Intercalating dyes typically bind andallow detection of any double stranded DNA. This is advantageous butthere is also the potential for binding to non-specific products andprimer dimers; careful PCR optimisation is required to determine theoptimum intercalating dye concentrations. The use of post-PCRapplications such as melting analysis can often distinguish betweena non-specific product/primer dimer and a PCR amplicon.

4.2.2. Potential issues with PCR-based approachesThe issue of PCR inhibition, due to DNA contaminants, potentially

causing false negatives needs to be taken into consideration for allPCR-based procedures (see below). Incorrect pipetting techniques oraerosol contamination of DNA and/or PCR products can contribute tofalse positives in all PCR-based applications. No Template Controls(NTC) should be used in every experiment to monitor PCR con-tamination, and laboratories should employ various decontaminationprocedures to avoid this (e.g. Ultraviolet sterilisation, decontamina-tion of bench tops).

5. Extraction of genomic DNA from environmental samples

Effective routine detection and surveillance of marine pests fromenvironmental samples requires effective sample collection and gDNAisolation methodology. Ophel-Keller et al. (2008) reported on theneed, when testing for soilborne diseases, to process gDNA from largesoil samples (N250 g–500 g), thus ensuring that the results are bio-logically relevant. Similar considerations need to be taken into ac-count when sampling sea and ballast water and sediments for marinepests. Organisms can be collected from water by filtering knownamounts and extracting DNA from the filtrate. Sediment samplesshould comprise approximately 500 g of sample, as for soilbornediseases (Ophel-Keller et al., 2008). Larger sample sizes are morelikely to contain pests.

Inhibition of PCR due to contaminants in environmental samplescannot be ignored. PCR inhibitors include; organic and phenolic com-pounds, fats, glycogen, Ca2+, humic acids, heavy metals, constituentsof bacterial cells, over-abundance of non-target DNA and other con-taminants (seeWilson, 1997). There is a need, therefore, to rigorouslyassess DNA extractions for the presence of PCR inhibitors to reduce theoccurrence of false negatives during analysis. Screening DNA extractswith control PCR assays of a known, added indicator organism is auseful approach in samples containing numerous DNA “types”. Correctpreparation of environmental samples for DNA extraction can alsoprove useful in preventing PCR inhibition. For example, freeze-dryingmarine sediment samples prior to DNA extraction greatly reduces theeffects of PCR inhibition (personal observations), which may be dueto increased DNA extraction efficiency in freeze-dried samples (asreported by Miller et al., 1999). Freeze-drying also helps to stabilizethe samplemaking it suitable for storage dry and at room temperature.A simple option to limit the effects of PCR inhibition is throughthe dilution of DNA; provided a reliable detection limit to detect anorganism is known then samples can be diluted to reduce inhibitorsto below critical concentrations.

6. Future approaches

Commercial shipping can act as a major vector in marine pesttranslocation (Barry et al., 2008). Ballast water commonly used tocontrol the trim and draft of a vessel can contain larval and adultstages of marine pest species (Minchin, 2007). The potential for thesepests to be discharged into a new environment and flourish is high forsome species (Hayes and Sliwa, 2003). Hull-fouling is also a significant

translocation vector, while solid ballast was a dominant vector inprevious centuries. As a consequence, ports are themajor entry pointsfor the majority of new marine pest incursions. Thus port and ballastwater surveys are targeted to areas where effective and efficientdetection and management strategies could be implemented to aidin successful prevention and mitigation of marine pest invasions.Whilst ports and ballast water are major entry points, the aquariumand aquaculture trade, specifically importation of exotic plants andanimals also needs to be monitored. The utilisation of marine pestmolecular detection technologies for these industries could improvemarine pest detection capabilities.

6.1. Routine monitoring and surveillance of marine pests in port waters

Routine monitoring using molecular technologies can alleviatethe demand for time-consuming and costly traditional port surveys.Understanding marine pest assemblages in ports is a vital tool formanaging ballast water in a cost-effective manner as themanagementrequirements are reduced or eliminated for transfers between portswith similar suites of pest species. Robust QPCR or microarrays,particularly in laboratories with high-throughput capability, can beused to rapidly assess samples for a wide range of pest species at afraction of the cost of traditional surveys. The vast majority of existingmolecular-based assays for marine pests (see Table 1) are not suitedto high-throughput applications so conversion of tests to formatssuch as QPCR is important. Dedicated research is required to providereliable, uniform testing procedures (e.g. QPCR orMicroarray) for pestspecies that display genetic differences across their range. Routinemonitoring programs utilising robust, sensitive and specific assaysfor a wide range of marine pest species providing the ability forauthorities to monitor and manage marine pest incursions in a cost-effective way.

6.2. Testing ballast water

The International Maritime Organisation (IMO) Convention forthe Control and Management of Ships' Ballast Water and Sediments(http://globallast.imo.org/resolution.htm) permits inspection ofships' ballast for harmful organisms (Article 9 Inspection of Ships)provided a ship is not unduly detained or delayed (Article 12 UndueDelay to Ships).Molecularmethods couldminimise delays and providedata on ballast water safety. There are technical and logistical hurdlesto provide suitable testing including timeliness to avoid unnecessarydelays. This is best achieved by streamlining sample collection andDNA extraction.

We propose that the most effective way to collect ballast watersamples is to filter a known amount of water (and ballast sediment)and collect the filtrate for subsequent DNA extraction. For this tooccur routinely, laboratory facilities may need to be maintained at, orclose to ports, and the DNA extraction method must not producePCR inhibition. Such processes may need to be developed includinglogistical, engineering and molecular biology methods to effectivelymonitor marine pests in ballast water. Well-defined quantitativetesting systems could also be used to test the performance of ballastwater treatment systems, particularly the concentrations of indicatormicrobes. However, the IMO Type Approval Guidelines do not cur-rently permit the use of molecular technologies to test treatmentsystems (http://globallast.imo.org/resolution.htm).

6.3. Future directions

Technology such as loop-mediated isothermal amplification pro-cedures (LAMP) (Notomi et al., 2000), nucleic acid sequence basedamplification (NASBA) (Compton, 1991), DNA detecting bioactivepaper (Ali et al., 2009) or linear after the exponential PCR (LATE-PCR)-based Smith's Detection (see Rice et al., 2007) all show potential for



use in “field-based” or “remote” testing. Multiplexed-tandem PCR(MT-PCR) has potential for use in remote laboratories (i.e. at or nearports) and can screen samples for a wide range of pest species at once(www.ausdiagnostics.com; Stanley and Szewczuk, 2005). The use ofSingle Nucleotide Polymorphism (SNP)-based assays (see Andrewand Kohn, 2009) for the detection and identification of marine pestspecies shows potential for accurate and rapid identification of closelyrelated species. The advent of testing facilities at or near ports wouldbetter enable the rapid turnaround required for effective screening ofballast water of commercial shipping.

The advent of next generation sequencing technologies, includingpyrosequencing-based 454 sequencing (www.454.com; Margulieset al., 2005), has revolutionised genomic research. It can sequenceentire genomes rapidly (Droege and Hill, 2008, Rothberg and Leamon,2008). 454 sequencing has also shown its utility in studies onmetagenomics (see Droege and Hill, 2008, Krause et al., 2006,Wommack et al., 2008). To date the use of next generation sequencinghas not been utilised for the detection of marine pests.

Sequencing by 454 technology has significant potential for futureuse in surveillance and detection of marine pests from environmentalsamples. Other next generation sequencing technologies such as theIllumina Genome Analyser (www.illumina.com, Bentley et al., 2008),ABI-SOLiD (www.appliedbiosystems.com, Pandey et al., 2008) andHelicos (www.helicosbio.com, Harris et al., 2008) also offer potentialfor future use in monitoring of environmental samples. The cost andbioinformatic processing associated with next generation sequencingtechnologies is currently prohibitive for regular surveillance andmonitoring of marine pests (see Rothberg and Leamon, 2008). If thesetechnologies become available to a wider range of laboratories andthe processing of bioinformatic data is streamlined, they may provevaluable for routine surveillance of different organisms from the samesample. The introduction of 454's desktop version, the GS Junior, maybring next generation sequencing technology to a wider range ofresearch laboratories, and enable this technology to be utilised for awider array of disciplines.

Methods that can screen environmental samples for a broad rangeof organisms at once, process large numbers of samples quickly, andprovide quantitative data, offer the ability to screen not only for pestspecies (or pathogens) but also for endemic species to provide a broaderindication of environmental health. Next generation sequencing tech-nology may also facilitate baseline screening of ports and harboursto obtain a rapid understanding of the presence and population den-sities of all species. This may aid in tailoring more focussed screeningfor marine pest species specific to a geographical area.

6.4. Legal requirements

It is possible that in the result of detection of marine pests throughmolecularmethods that legal action (e.g. seizures, quarantine)wouldbeinitiated by the relevant jurisdiction. It is therefore of utmost impor-tance that positive results are verified through a number of methods:1. manual discovery and identification of the pest; 2. obtaining DNAsequence data from the sample to further confirm identity; 3. usingmultiple detection methods. It is also essential that the laboratoryinvolved follow rigorous protocols and utilise a number of controls(including spikes) to provide quality control and assurance measures.

7. Concluding remarks

This review summarises studies about the molecular identifica-tion and/or detection of marine pest species. For effective control ofmarine pest incursions, rapid specific identification is of utmost im-portance. An understanding and exploration of the genetic diversityof different pest species populations is also required, which will leadto more effective control and a better understanding of pest biology.The development of molecular based assays for a wide range of

marine pests provides a solid foundation for the continued develop-ment of robust, sensitive and specific assays for screening environ-mental samples. It is important that the opportunity is grasped todevelop environmental testing facilities for marine pests, as in somesituations detection may prevent release unless appropriate treat-ment is undertaken. Identification is vital in order for treatment andmitigation programmes to be implemented in a timely and effectivemanner.

As a practical first step, assays should be developed for use indedicated research or government diagnostic facilities. Provided thatthey are robustly validated, QPCR currently offers the most cost-effective and rapid means of analysis. In order to develop rapid testingcapabilities, the ability to carry out testing at the point of collection(e.g. LAMP, NASBA, LATE-PCR, MT-PCR) may need to be consideredand evaluated in the future. The potential of metagenomics/nextgeneration sequencing applications cannot be ignored, with the ad-vent of improved genome sequencing (e.g. 454, Illumina, ABI-SOLiD,Helicos) providing opportunities to not only understand what pestspecies are present in a sample but also to analyse endemic species toassess environmental health.

An important component in the development of assays is an ef-fective DNA isolation and purification technique thatworks effectivelyon a range of sample types (e.g. sediments and water). Methods willneed to be optimised to remove inhibitory substances to enzymaticamplification and development of an accepted standard for the ef-fective purification of DNA from different sample types is essential.

Continued development of PCR-based marine pest assays mustoccur in conjunction with taxonomic specialists to enable a thoroughunderstanding of species and/or strains. Studies investigating thesystematics and phylogenetic relationships of marine pest species andrelated taxa will progress towards the development of robust di-agnostic markers. These studies will help to further identify molecularmarkers with diagnostic potential, and provide baseline genetic in-formation necessary for the design of specific diagnostic assays.

Specific diagnosis is central to: (a) rapidly establishing the prev-alence and distribution of marine pest species in the environment inconjunction with traditional sampling techniques; (b) monitoringchanges in marine pest distribution spatially and temporally; and(c) conducting targeted eradication and control programmes if eco-nomics and logistics permit. Developing the capacity for detection andenumeration of marine pest species seems achievable if appropriatesupport and funding is provided by relevant stakeholders.

Acknowledgements

The authors would like to thank the Australian Department ofEnvironment, Water, Heritage and Arts, Primary Industries andResources - South Australia, and the Adelaide andMount Lofty RangesNatural Resources Management Board for funding. NJB, KPR, JET andMRD are supported by Marine Innovation South Australia (MISA), aninitiative of the South Australian Government. This research formspart of the work of the MISA Biosecurity Node.

References

Afonina IA, Reed MW, Lusby E, Shishkina IG, Belousov YS. Minor groove binderconjugated DNA probes for quantitative DNA detection by hybridization-triggeredfluorescence. Biotechniques 2002;32:940–9.

Ahn S, Kulis DM, Erdner DL, Anderson DM, Walt DR. Fiber-optic for the simultaneousdetection of multiple harmful algal bloom species. Appl Environ Micro 2006;72:5742–9.

Ali MM, Aguirre SD, Xu Y, Filipe CDM, Pelton R, Li Y. Detection of DNA using bioactivepaper strips. Chem Comm 2009;43:6640–2.

Anderson LWJ. California's reaction to Caulerpa taxifolia: a model for invasive speciesrapid response. Biol Inv 2005;7:1003–16.

Anderson DM, Kulis DM, Keafer BA, Gribble KE, Marin R, Scholin CA. Deep Sea Res II2005;52:2467–90.

Andrew M, Kohn LM. Single Nucleotide Polymorphism-based diagnostic system forcrop-associated Sclerotinia species. Appl Env Micro 2009;75:5600–6.



Ayers K, Rhodes LL, Tyrell J, Gladstone M, Scholin C. International accreditation ofsandwich hybridisation assay format DNA probes for micro-algae. NZ J Mar FreshRes 2005;39:1225–31.

Baldwin BS, Black M, Sanjur O, Gustafson R, Lutz RA, Vrijenhoek RC. A diagnosticmolecular marker for zebra mussels (Dreissena polymorpha) and potentially co-occurring bivalves:mitochondrial COI. Mol Mar Bio Biotech 1996;5:9-14.

Barry SC, Hayes KR, Hewitt CL, Behrens HL, Dragsund E, Bakke SM. Ballast water riskassessment: principles, processes, and methods. ICES J Mar Sci 2008;65:121–31.

Bax N, Williamson A, Aguero M, Gonzalez E, Geeves W. Marine invasive alien species: athreat to global biodiversity. Mar Pol 2003;27:313–23.

Becker A, Reith A, Napiwotzki J, Kadenbach B. A quantitative method of determininginitial amounts of DNA by polymerase chain reaction cycle titration using digitalimaging and a novel DNA stain. Anal Biochem 1996;237:204–7.

Bentley DR, Balasubramanian S, Swerdlow HP, Smith GP, Milton J, et al. Accurate wholehuman genome sequencing using reversible terminator chemistry. Nature 2008;456:53–9.

Blair D, Waycott M, Byrne L, Dunshea G, Smith-Keune C, Neil KM. Moleculardiscrimination of Perna (Mollusca: Bivalvia) species using the polymerase chainreaction and species-specific mitochondrial primers. Mar Biotech 2006;8:380–5.

Blomster J, Hoey EM, Maggs CA, Stanhope MJ. Species-specific oligonucleotide probesfor macroalgae: molecular discrimination of two marine fouling species ofEnteromorpha (Ulvophyceae). Mol Ecol 2000;9:177–86.

Boeger WA, Pie MR, Falleros RM, Ostrensky A, Darrigan G, Mansur MCD, et al. Testing amolecular protocol to monitor the presence of golden mussel larvae (Limnopernafortunei) in plankton samples. J Plank Res 2007;29:1015–9.

Booth D, Proven J, Maggs CA. Molecular approaches to the study of invasive seaweeds.Bot Mar 2007;50:385–96.

Bott NJ, Campbell BE, Beveridge I, Chilton NB, Rees D, Hunt PW, et al. A combinedmicroscopic-molecular method for the diagnosis of strongylid infections in sheep.Int J Para 2009;39:1277–87.

Bowers HA, Tengs T, Glasgow Jr HB, Burkholder JM, Rublee PA, Oldach DW.Development of real-time PCR assays for the rapid detection of Pfiesteria piscicidaand related dinoflagellates. Appl Env Micro 2000;66:4641–8.

Buonoccorsi VP, Starkey E, Graves JE. Mitochondrial and nuclear DNA analysis ofpopulation subdivision among young-of-the-year Spanish mackerel (Scomberomorusmaculates) from the western Atlantic and Gulf of Mexico. Mar Biol 2001;138:37–45.

Campbell ML, Gould B, Hewitt CL. Survey evaluations to assess marine bioinvasions.Mar Poll Bull 2007;55:360–78.

Chen X, Kwok PY. Homogeneous genotyping assays for single nucleotide polymorphismswith fluorescence resonance energy transfer detection. Genet Anal 1999;14:157–63.

Chu KH, Ho HY, Li CP, Chan TY. Molecular phylogenetics of the mitten crab species inEriocheir, sensu lato (Brachyura: Grapsidae). J Crus Biol 2003;23:738–46.

ClaxtonWT, Boulding EG. A newmolecular technique for identifying field collections ofzebra mussel (Dreissena polymorpha) and quagga mussel (Dreissena bugensis)veliger larvae applied to eastern Lake Erie, Lake Ontario, and Lake Simcoe. Can JZool 1998;76:194–8.

Clegg MT, Gaut BS, Learn GH, Morton BR. Rates and patterns of chloroplast DNAevolution. PNAS 1994;91:6795–801.

Cohen AN, Carlton JT. Accelerating invasion rate in a highly invaded estuary. Science1998;279:555–8.

Compton J. Nucleic acid sequence-based amplification. Nature 1991;350:91–2.Crombie J, Knight E, Barry S. Marine pest incursions- a tool to predict the cost of eradication

basedonexpert assessments. Canberra, Australia: Bureauof Rural Services; 2007. 61pp.Cross I, Rebordinos L, Diaz E. Species identification of Crassostrea and Ostrea oysters by

polymerase chain reaction amplification of the 5S rRNA gene. J AOAC Int 2006;89:144–8.

Deagle BE, Bax N, Hewitt CL, Patil JG. Development and evaluation of a PCR-based testfor detection of Asterias (Echinodermata: Asteroidea) larvae in Australian planktonsamples from ballast water. Mar Fresh Res 2003;54:709–19.

Diercks S, Metfies K, Medlin LK. Development and adaption of a mulitprobe biosensorfor the use in a semi-automated device for the detection of toxic algae. BiosensBioelectron 2008;23:1527–33.

DroegeM,Hill B. TheGenome Sequencer FLXTM system-Longer reads,more applications,straight forward bioinformatics and more complete data sets. J Biotech 2008;136:3-10.

Eickbush TH, Eickbush DG. Finely orchestrated movements: evolution of the ribosomalRNA genes. Genetics 2007;175:477–85.

Elder JF, Turner BJ. Concerted evolution of repetitive DNA sequences in eukaryotes.Q Rev Biol 1995;70:297–320.

Espy MJ, Uhl JR, Sloan LM, Buckwater SP, Jones MF, et al. Real-time PCR in clinicalmicrobiology: applications for routine laboratory testing. ClinMicroReviews2006;19:165–256.

Fama P, Jousson O, Zaninetti L, Meinesz A, Dini F, Di Giuseppe G, et al. Geneticpolymorphism in Caulerpa taxifolia (Ulvophyceae) chloroplast DNA revealed by aPCR-based assay of the invasive Mediterranean strain. J Evol Biol 2002;15:618–24.

Fernandez-Tajes J, Mendez J. Identification of the Razor clam species Ensis arcuatus, E.siliqua, E. directus, E. macha, and Solen marginatus using PCR-RFLP analysis of the 5SrDNA region. J Agric Food Chem 2007;55:7278–82.

Freshwater DW, Montgomery F, Greene JK, Hamner RM, Williams M, Whitfield PE.Distribution and identification of an invasive Gracilaria speciesthat is hamperingcommercial fishing operationsin southeastern North Carolina, USA. Biol Inv 2006;8:631–7.

Frischer ME, Hansen AS, Wyllie JA, Wimbush J, Murray J, Nierwicki-Bauer SA. Specificamplification of the 18S rRNA gene as a method to detect zebra mussel (Dreissenapolymorpha) larvae in plankton samples. Hydrobiologia 2002;487:33–44.

Galil BS. Loss or gain? Invasive aliens and biodiversity in the Mediterranean Sea. MarPoll Bull 2007;55:314–22.

Galil BS, Bogi C. Mytilopsis sallei (Mollusca: Bivalvia: Dreissenidae) established on theMediterranean coast of Israel. Mar Bio Rec; 2: e73,1-4.

Galluzzi L, Penna A, Bertozzini E, Vila M, Garcés E, Magnani M. Development of a real-time PCR assay for rapid detection and quantification of Alexandrium minutum (adinoflagellate). Appl Env Micro 2004;70:1199–206.

Gasser RB, Hu M, Chilton NB, Campbell BE, Jex AJ, Otranto D, et al. Single-strandedconformation polymorphism (SSCP) for the analysis of genetic variation. Nat Protoc2006;1:3121–8.

Godhe A, Otta SK, Rehnstam-Holm A-S, Karunasagar I, Karunasagar I. Polymerase chainreaction in detection of Gymnodinium mikimotoi and Alexandrium minutum in fieldsamples from southwest India. Mar Biotech 2001;3:152–62.

Godhe A, Cusack C, Pederson J, Anderson P, Anderson DM, et al. Intercalibration ofclassical and molecular techniques for identification of Alexandrium fundyense(Dinophyceae) and estimation of cell densities. Harm Alg 2007;6:56–72.

Goffredi SK, Jones WJ, Scholin CA, Marin III R, Vrijenhoek RC. Molecular detection ofmarine invertebrate larvae. Mar Biotech 2006;8:149–60.

Golenberg EM, Clegg MT, Durbin ML, Doebley J, Ma DP. Evolution of a noncoding regionof the chloroplast genome. Mol Phyl Evo 1993;2:52–64.

Gunasekera RM, Patil JG, McEnnulty FR, Bax NJ. Specific amplification of mt-CO1 gene ofthe invasive gastropod Maoricolpus roseus in planktonic samples reveals a free-living larval life-history stage. Mar Fresh Res 2005;56:901–12.

Hanyuda T, Arai S, Ueda K. Variability in the rbcL introns of Caulerpalean algae(Chlorphyta, Ulvophyceae). J Plant Res 2000;113:403–13.

Hare MP, Palumbi SR, Butman CA. Single-step species identification of bivalve larvaeusing multiplex polymerase chain reaction. Mar Biol 2000;137:953–61.

Harris TD, Buzby PR, Babcock H, Beer E, Bowers J, et al. Single-molecule DNA sequencingof a viral genome. Science 2008;320:106–9.

Harrison RG. Animal mitochondrial DNA as a genetic marker in population andevolutionary biology. Tree 1989;4:6-11.

Harvey JBJ, Hoy MS, Rodriguez RJ. Molecular detection of native and invasive marineinvertebrate larvae present in ballast and open water environmental samplescollected in Puget Sound. J Exp Mar Bio Eco 2009;369:93–9.

Hayes KR, Sliwa C. Identifying potential marine pests- a deductive approach applied toAustralia. Mar Poll Bull 2003;46:91–8.

Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res1996;6:986–94.

Higuchi R, Fockler C, Dollinger G, Watson R. Kinetic PCR analysis: real-time monitoringof DNA amplification reactions. Biotechnology (NY) 1993;11:1026–30.

Hillis DM, Dixon MT. Ribosomal DNA: molecular evolution and phylogenetic inference.Q Rev Biol 1991;66:411–53.

Hopkins GW, Freckleton RP. Declines in numbers of amateur and professional taxonomists:implications for conservation. Anim Conserv 2002;5:245–9.

Jones WJ, Preston CM, Marin III R, Scholin CA, Vrijenhoek RC. A robotic molecularmethod for in situ detection of marine invertebrate larvae. Mol Ecol Res 2008;8:540–50.

Kamikawa R, Hosoi-Tanabe S, Yoshimatsu S, Oyama K,Masuda I, Sako Y. Development of anovel molecular marker on the mitochondrial genome of a toxic dinoflagellate,Alexandrium spp., and its application in single-cell PCR. J Appl Phycol 2008;20:153–9.

Kawakami T, Butlin RK, Adams M, Saint KM, Paull DJ, Cooper SJB. Differential geneflow of mitochondrial and nuclear DNA markers among chromosomal races ofAustralian morabine grasshoppers (Vandiemenella, viatica species group). Mol Ecol2007;16:5044–56.

Ki JS, Han MS. A low-density oligonucleotide array study for the parallel detection ofharmful algal species using hybridization of consensus PCR products of LSU rDNAD2 domain. Biosens Bioelectron 2006;21:1812–21.

Kim KC, Byrne LB. Biodiversity loss and the taxonomic bottleneck: emergingbiodiversity science. Ecol Res 2006;21:794–810.

Kocher TD, Thomas WK, Meyer A, Edwards SV, Pāābo S, Villablanca FX, et al. Dynamicsof mitochondrial DNA evolution in animals: amplification and sequencing withconserved primers. PNAS 1989;86:6196–200.

Krause L, Diaz NN, Bartels D, Edwards RA, Pühler A, Rohwer F, et al. Finding novel genesin bacterial communities isolated from the environment. Bioinformatics 2006;22:e281–9.

Larsen JB, Frischer ME, Rasmussen LJ, Hansen BW. Single-step nested multiplex PCR todifferentiate between various bivalve larvae. Mar Biol 2005;146:1119–29.

Larsen JB, FrischerME, OckelmannKW, Rasmussen LJ, Hansen BW. Temporal occurrenceof planktotrophic bivalve larvae identified morphologically and by single stepnested multiplex PCR. J Plank Res 2007;29:423–36.

Lazerges M, Perrot H, Antoine E, Defontaine A, Compere C. Oligonucleotide quartzcrystal microbalance sensor for the microalgae Alexandrium minutum (Dinophy-ceae). Biosens Bioeclectron 2006;21:1355–8.

Le Goff-Vitry MC, Chipman AD, Comtet T. In situ hybridization on whole larvae: a novelmethod for monitoring bivalve larvae. Mar Ecol Prog Ser 2007;343:161–72.

Livi S, Cordisco C, Damiani C, Romanelli M, Crosetti D. Identification of bivalve species atan early developmental stage through PCR-SSCP and sequence analysis of partial18S rDNA. Mar Biol 2006;149:1149–61.

Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, et al. Genome sequencing inopen microfabricated high density picoliter reactors. Nature 2005;437:376–80.

Mbwana J, Bölin I, Lyamuya E, Mhalu F, Lagergård T. Molecular characterization ofHaemophilus ducreyi isolates from different geographical locations. J Clin Micro2006;44:132–7.

Miller DN, Bryant JE, Madsen EL, Ghiorse WC. Evaluation and optimization of DNAextraction and purification procedures for soil and sediment samples. Appl EnvMicro 1999;65:4715–24.



Minchin D. Aquaculture and transport in a changing environment: overlap and links inthe spread of alien biota. Mar Poll Bull 2007;55:302–13.

Monis PT, Giglio S, Saint CP. Comparison of SYTO9 and SYBR Green I for real-timepolymerase chain reaction and investigation of the effect of dye concentration onamplification and DNA melting curve analysis. Anal Biochem 2005;340:24–34.

Morgan TS, Rogers AD. Specificity and sensitivity of microsatellite markers for theidentification of larvae. Mar Biol 2001;139:967–73.

Morton BR. Neighboring base composition and transversion/transition bias in acomparison of rice and maize chloroplast noncoding regions. PNAS 1995;92:9717–21.

Mountfort D, Rhodes L, Broom J, Gladstone M, Tyrell J. Fluorescent in situ hybridisationassay as a species-specifc identifier of the northern Pacific seastar, Asterias amurensis.NZ J Mar Fresh Res 2007;41:283–90.

Mullis KB, Faloona F, Scharf S, Saiki R, Horn G, Erlich H. Specific enzymatic amplificationof DNA in vitro: the polymerase chain reaction. Cold Spring Harbor Symp QuantBiol 1986;51:263–73.

NIMPCG. Australian Marine Pest Monitoring Guidelines Version 2.0. 2009a. Departmentof Agriculture, Fisheries and Forestry (DAFF). Ed. National Introduced Marine PestsCoordination Group. 48 pp.

NIMPCG.Marine PestMonitoringManual Version 2.0. 2009b. Department of Agriculture,Fisheries and Forestry (DAFF). Ed. National Introduced Marine Pests CoordinationGroup. 92 pp.

Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 2000;28:e63.

Ophel-Keller K, McKay A, Hartley D. Herdina. Curran J. Development of a routine DNA-based testing service for soilborne diseases in Australia. Aust Plant Path 2008;37:243–53.

Palmer JD. Contrastingmodes and tempos of genome evolution in land plant organelles.Trends Genet 1990;6:115–20.

PanM,McBeath AJA, Hay SJ, Pierce GJ, Cunnigham CO. Real-time PCR assay for detectionand relative quantification of Liocarcinus depurator larvae from plankton samples.Mar Biol 2008;153:859–70.

Pandey V, Nutter RC, Prediger E. Applied Biosystems SOLiD™ System: ligation-basedsequencing, next generation genome sequencing: towards personal medicine.Wiley;2008. p. 29–41.

Pangasa A, Jex AR, Campbell BE, Bott NJ, Whipp M, Hogg G, et al. High resolutionmelting-curve (HRM) analysis for the diagnosis of cryptosporidiosis in humans.Mol Cell Probes 2009;23:10–5.

Park TG, de Salas MF, Bolch CJS, Hallegraff GM. Development of a Real-time PCR probefor quantification of the heterotrophic dinoflagellate Cryptoperidinopsis brodyi(Dinophyceae). Appl Env Micro 2007;73:2552–60.

Patil JG, Gunasekera RM, Deagle BE, Bax NJ, Blackburn SI. Development and evaluationof a PCR based assay for detection of the toxic dinoflagellate, Gymnodiniumcatenatum (Graham) in ballast water and environmental samples. Biol Inv 2005a;7:983–94.

Patil JG, Gunasekera RM, Deagle BE, Bax NJ. Specific detection of Pacific oyster (Crassostreagigas) larvae inplankton samplesusing nested polymerase chain reaction.Mar Biotech2005b;7:11–20.

Penna A, Magnani M. Identification of Alexandrium (Dinophyceae) species using PCRand rDNA-targetted probes. J Phycol 1999;35:615–21.

Piatek AS, Tyagi S, Pol AC, Telenti A, Miller LP, Kramer FR, et al. Molecular beaconsequence analysis for detecting drug resistance in Mycobacterium tuberculosis. NatBiotechnol 1998;16:359–63.

PieMR, BoegerWA, Patella L, Falleiros RM. A fast and accurate molecular method for thedetection of larvae of the golden mussel Limnoperna fortunei (Mollusca: Mytilidae)in plankton samples. J Moll Stud 2006;72:218–9.

Pradillon F, Schmidt A, Peplies J, Dubilier N. Species identification of marineinvertebrate early stages by whole-larvae in situ hybridisation of 18S ribosomalRNA. Mar Ecol Prog Ser 2007;333:103–16.

Rand DM. Thermal habit, metabolic rate and the evolution of mitochondrial DNA. TREE1994;9:125–31.

Rice JE, Sanchez JA, Pierce KE, Reis Jr AH, Osborne A, Wangh LJ. Monoplex/multiplexlinear-after-the-exponential-PCR assays combined with Primesafe and Dilute-‘N’-Go sequencing. Nat Protoc 2007;2:2429–38.

Rothberg JM, Leamon JH. The development and impact of 454 sequencing. Nat Biotech.2008;26:1117–24.

Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA.Primer-directed enzymatic amplification of DNA with a thermostable DNApolymerase. Science 1988;239:487–91.

Scholin CA, Anderson DM. Identification of group-specific and strain-specific genetic-markers for globally distributed Alexandrium (Dinophyceae). 1. RFLP analysis ofSSU ribosomal-RNA genes. J. Phycol 1994;30:744–54.

Soria-Hernanz DF, Braverman JM, Hamilton MB. Parallel rate heterogeneity inchloroplast and mitochondrial genomes of Brazil nut trees (Lecythidaceae) isconsistent with lineage effects. Mol Biol Evol 2008;25:1282–96.

Stanley KK, Szewczuk E. Multiplexed tandem PCR: gene profiling from small amounts ofRNA using SYBR Green detection. Nuc Acids Res 2005;33:e180.

Stepien CA, Hubers AN, Skidmore JL. Diagnostic genetic markers and evolutionaryrelationships among invasive dreissenoid and corbiculoid Bivalves in North America:phylogenetic signal frommitochondrial 16S rDNA. Mol Phy Evo 1999;13:31–49.

Takahashi Y, Takishita K, Koike K, Maruyama T, Nakayama T, Kobiyama A, et al.Development of molecular probes for Dinophysis (Dinophyceae) plastid: a tool topredict blooming and explore plastid origin. Mar Biotech 2005;7:95-103.

Tang B, Zhou K, Song D, Yang G, Dai A. Molecular systematics of the Asian mitten crabs,genus Eriocheir (Crustacea: Brachyura). Mol Phyl Evo 2003;29:309–16.

Touzet N, Raine R. Discrimination of Alexandrium andersoni and A. minutum(Dinophyceae) using LSU rDNA-targeted oligonucleotide probes and fluorescentwhole-cell hybridization. Phycologia 2007;46:168–77.

Tyrell JV, Connell LB, Scholin CA. Monitoring of Heterosigma akashiwo using a sandwichhybridization assay. Harmful Algae 2002;1:205–14.

Wallentinus I, Nyberg CD. Introducedmarine organisms as habitat modifiers. Mar PollutBull 2007;55:323–32.

Wang S, Bao Z, Zhang L, Li N, Zhan A, Guo W, et al. A new strategy for speciesidentification of planktonic larvae: PCR–RFLP analysis of the internal transcribedspacer region of ribosomal DNA detected by agarose gel electrophoresis or DHPLC.J Plank Res 2006a;28:375–84.

Wang W, Chen K, Xu C. DNA quantification using EvaGreen and a real-time PCRinstrument. Anal Biochem 2006b;356:303–5.

Wilson IG. Inhibition and facilitation of nucleic acid amplification. Appl Environ Micro1997;63:3741–51.

Wittwer CT, Reed GH, Gundry CN, Vandersteen JG, Pryor RJ. High-resolution genotypingby amplicon melting analysis using LCGreen. Clin Chem 2003;49:853–60.

Wommack KE, Bhavsar J, Ravel J. Metagenomics: read length matters. Appl EnvironMicro 2008;74:1453–63.

Xu H, DeLuca SZ, O'Farrell PH. Manipulation of the metazoan mitochondrial genomewith targeted restriction enzymes. Science 2008;321:575–7.

Yamasaki I, Yoshizaki G, Yokota M, Strüssmann CA, Watanabe S. Mitochondrial DNAvariation and population structure of the Japanese mitten crab Eriocheir japonica.Fish Sci 2006;72:299–309.

Zhou KY, Gao ZQ. Identification of the mitten crab Eriocheir sinensis populations usingRAPD markers. Chi J App Env Bio 1999;5:176–80.


Toward routine, DNA-based detection methods for marine pests

Documents

Transcript of Toward routine, DNA-based detection methods for marine pests