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An improved real-time PCR assay for the detection of Old World screwworm flies Jess A.T. Morgan a,∗ , Rudolf Urech b a b

Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St Lucia, Queensland, Australia Department of Agriculture, Forestry and Fisheries, Eco-Sciences Precinct, Queensland, Australia

a r t i c l e

i n f o

Article history: Received 17 October 2013 Received in revised form 21 February 2014 Accepted 25 February 2014 Available online xxx Keywords: Chrysomya bezziana Myiasis IAC Diagnostics mtDNA Multiplex

a b s t r a c t The Old World screwworm (OWS) fly, Chrysomya bezziana, is a serious pest of livestock, wildlife and humans in tropical Africa, parts of the Middle East, the Indian subcontinent, south-east Asia and Papua New Guinea. Although to date Australia remains free of OWS flies, an incursion would have serious economic and animal welfare implications. For these reasons Australia has an OWS fly preparedness plan including OWS fly surveillance with fly traps. The recent development of an improved OWS fly trap and synthetic attractant and a specific and sensitive real-time PCR molecular assay for the detection of OWS flies in trap catches has improved Australia’s OWS fly surveillance capabilities. Because all Australian trap samples gave negative results in the PCR assay, it was deemed necessary to include a positive control mechanism to ensure that fly DNA was being successfully extracted and amplified and to guard against false negative results. A new non-competitive internal amplification control (IAC) has been developed that can be used in conjunction with the OWS fly PCR assay in a multiplexed single-tube reaction. The multiplexed assay provides an indicator of the performance of DNA extraction and amplification without greatly increasing labour or reagent costs. The fly IAC targets a region of the ribosomal 16S mitochondrial DNA which is conserved across at least six genera of commonly trapped flies. Compared to the OWS fly assay alone, the multiplexed OWS fly and fly IAC assay displayed no loss in sensitivity or specificity for OWS fly detection. The multiplexed OWS fly and fly IAC assay provides greater confidence for trap catch samples returning negative OWS fly results. © 2014 International Atomic Energy Agency. Published by Elsevier B.V. All rights reserved.

1. Introduction The Old World screwworm (OWS) fly, Chrysomya bezziana Villeneuve, (Diptera: Calliphoridae), is a serious pest of livestock, wildlife and humans in tropical Africa, the Indian subcontinent, parts of the Middle East, south-east Asia including the Indonesian and Philippine islands and Papua New Guinea (Spradbery, 1994; World Organization for Animal Health (OIE), 2013). Endemic populations of OWS flies have been found at relatively high densities along the Torres Strait coast in Papua New Guinea, just 150 km from mainland Australia (Spradbery and Tozer, 2013). Although to date Australia remains free of screwworm flies, the government and livestock industries are aware of the economic and welfare consequences of an OWS fly incursion. A preparedness plan (AUSVETPLAN, 2007) is in place which includes surveillance for adult and immature flies, awareness

∗ Corresponding author. Tel.: +61 7 3255 4527; fax: +61 7 3346 2167. E-mail address: [email protected] (J.A.T. Morgan).

campaigns, a bioeconomic model (Anaman et al., 1993, 1994) and plans for containment and subsequent eradication of OWS flies by sterile insect technology in case of an incursion. The risks of entry of OWS flies into Australia and surveillance requirements are currently being reviewed (Animal Health Australia, 2012). The development of an improved model for managing OWS fly incursions into Australia is nearing completion. The new model is agent (fly) based and incorporates up-to-date environmental parameters (Welch et al., ref. in this issue). Surveillance for adult OWS flies using fly traps is conducted in areas considered to be at high risk of incursion, currently on four Torres Strait Islands and around livestock shipping ports in Australia. Improved OWS fly traps and synthetic attractant were developed and field tested on cattle farms in Malaysia by Urech et al. (2012). The modified LuciTrap® with Bezzilure-2 attractant caught on average 3.1 times more OWS flies than the standard sticky trap using Swormlure (Urech et al., 2012). The traps also provided selectivity for C. bezziana against other blowfly species (selectivity factors of 3.6–90 against common non-target species). Despite

http://dx.doi.org/10.1016/j.actatropica.2014.02.015 0001-706X/© 2014 International Atomic Energy Agency. Published by Elsevier B.V. All rights reserved.

Please cite this article in press as: Morgan, J.A.T., Urech, R., An improved real-time PCR assay for the detection of Old World screwworm flies. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.02.015

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the traps’ increased specificity and selectivity, there was a significant by-catch with C. bezziana comprising about 10% of the total fly catch. In addition the modified LuciTraps® are capable of holding more than 50,000 flies, vastly more than sticky traps that have a saturation capacity of 1500–2000 flies (Urech et al., 2012) and by-catch remain difficult to differentiate. A DNA extraction protocol to extract up to 1000 flies, and a C. bezziana-specific real-time PCR molecular assay were developed by Jarrett et al. (2010). The assay uses species-specific primers and a C. bezziana-specific Taqman® MGB (minor groove binding) probe that target the multicopy ribosomal DNA internal transcribed spacer 1 region. The assay was found to be sensitive enough to detect one C. bezziana in a sample of 1000 non-target flies (Jarrett et al., 2010). When used in combination with the improved attractant and trap, the real-time PCR assay greatly increases OWS fly screening capabilities in surveillance areas where prevalence is low. The assay successfully amplified OWS flies from Indonesia, Malaysia, Papua New Guinea, India, Africa and the UAE (Jarrett et al., 2010) and has since amplified OWS flies from Iraq (authors, unpublished). Biosecurity Australia (formerly the Australian Quarantine Inspection Service) was keen to adopt the real-time PCR molecular assay for testing large trap catches collected as part of their surveillance program. Screening using the molecular assay, using appropriate negative (water) and positive (OWS fly DNA) controls, has thus far returned only negative (no amplification) results for Australian trap samples, confirming the results from morphological inspections. However, false-negative results can arise from PCR inhibition, target DNA degradation, sample processing errors and thermal cycler malfunction (Hoorfar et al., 2004). The real-time PCR assay lacked an internal amplification control (IAC) to confirm that DNA was being successfully extracted and that PCR inhibitors were not blocking OWS fly DNA amplification. IACs provide an accurate way to assess the integrity of all the steps in a nucleic acid amplification assay (Nolte, 2004). Early OWS fly detection will be critical for successful containment and eradication (Maynard et al., 2004) and an IAC was deemed necessary to lower the probability of false negatives and potential delays in detection (Rosenstraus et al., 1998). The mitochondrial genome is commonly targeted for DNAbased identification of flies (Wells and Sperling, 2001; Wallman and Donnellan, 2001; Li et al., 2010; Guo et al., 2011). A region of the mitochondrial DNA 16S gene was targeted for the IAC because this gene is functionally important and contains conserved sequence domains (Simon et al., 1994). Primers were designed to match regions of sequence in this multi-copy target that were conserved among fly families as diverse as the Calliphoridae, Oestridae, Drosophilidae, Tachinidae, Muscidae and Sarcophagidae. This broad level of coverage spans the majority of fly species caught in central indo-pacific fly traps (Urech et al., 2008, 2012). The conserved fly mitochondrial DNA is co-amplified simultaneously with the target C. bezziana nuclear DNA in a one-tube assay. In a PCR containing the IAC, a fly signal should be produced even if there is no C. bezziana present. This paper reports the development and evaluation of a positive fly control real-time PCR assay that can be used in conjunction with the assay developed by Jarrett et al. (2010) in a multiplexed singletube reaction.

2. Materials and methods 2.1. Fly genomic DNA DNA extractions from individual fly species were those used in Jarrett et al. (2010) (Table 1). DNA from field and laboratory samples were those processed in Morgan et al. (2008) plus additional trap

Table 1 Fly species used to test the fly IAC real-time PCR assay. Fly tissue

Origin

Source

Chrysomya bezziana C. bezziana C. bezziana C. flavifrons C. incisuralis C. latifrons C. megacephala C. megacephala C. nigripes C. putoria C. rufifacies C. saffranea C. semimetallica C. varipes Cochliomyia hominivorax Hemipyrellia sp. Lucilia cuprina Musca domestica Sarcophagidae

Lab Lab Lab Field Field Field Lab Field Field Field Lab Field Field Field Lab Field Lab Lab Field

Bogor, Java, Indonesia East Sumba, Indonesia Maros, Indonesia Kuranda, Queensland, AUS Kuranda, Queensland, AUS Mt Keira, NSW, AUS Yeerongpilly, Queensland, AUS Malaysia Kuranda, Queensland, AUS Grahamstown, South Africa Longreach, Queensland, AUS Yeerongpilly, Queensland, AUS Kuranda, Queensland, AUS Kuranda, Queensland, AUS Mexico Yeerongpilly, Queensland, AUS Canberra, ACT, AUS Novartis laboratory culture Yeerongpilly, Queensland, AUS

samples as specified below. Briefly, for amplification consistency and preliminary assay testing, DNA that had been extracted from a uniform starting material consisting of four replicate samples of 1 C. bezziana (laboratory colony from Bogor, Java, Indonesia) plus 999 C. megacephala (laboratory colony from Yeerongpilly, Queensland, Australia) were used. These replicates provide an indication of the sample to sample variability in CT scores obtained from DNA extractions of large numbers of flies. The IAC assay was then tested against field trap catches of varying sizes (21–1020 flies) from Malaysia that were known to contain from one to five C. bezziana. Surveillance trap catches from the Torres Strait Islands which, based on morphological examination, were known to be C. bezziana-free were also tested. 2.2. Real-time PCR assay design To ensure generic amplification of fly DNA in the real-time PCR assay a sequence alignment was constructed for a range of fly genera and species (Table 2). The forward primer, reverse primer and fluorogenic probe were designed in a region of 16S mitochondrial ribosomal DNA sequence that was 100% conserved across all of the species (Table 3). Primers were designed to be 20–30 bp long with similar melting temperatures (Tm ) above 60 ◦ C and G + C nucleotide content between 20 and 80%. No more than three G and or C nucleotides were permitted in the last 5 positions at the 3 end of the primer to reduce non-specific priming. The length of the amplification product was kept under 200 bp. The Taqman® MGB probe was designed with a Tm of 10 ◦ C higher than the primers and GC content between 30 and 80%. The probe was labelled with a VIC fluorophore to enable a multiplex detection assay with the C. bezziana-specific assay developed by Jarrett et al. (2010). Real-time PCR assays were conducted on a Rotor-Gene 3000 (Corbett Research, Qiagen, Victoria, Australia). Initial reactions were run in a total volume of 20 ␮l containing 8 ␮l RealMasterMix Probe (Eppendorf, NSW, Australia), IAC PCR primers FLY16SF and FLY16SR (alone or multiplexed with the OWS fly assay primers RTBezF1 and RTBezR1) at a concentration of 300 nM each, the IAC Taqman® MGB probe FLY16S VIC PROBE (Minor Groove Binding probe Applied Biosystems, New York, USA) (alone or multiplexed with the OWS fly assay probe BezFAM) at 200 nM each and 5 ␮l of genomic DNA (concentrations ranging over 3 orders of magnitude). A negative control that replaced DNA with milliQ water was run alongside all samples. Amplification conditions were 2 min at 95 ◦ C followed by 45 cycles of 15 s at 95 ◦ C, 20 s at 60 ◦ C and 20 s at 68 ◦ C, acquiring FAM and JOE (VIC Channel) at the end of this

Please cite this article in press as: Morgan, J.A.T., Urech, R., An improved real-time PCR assay for the detection of Old World screwworm flies. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.02.015

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Species

JX913760 JX913736 JX913737 JX913738 JX913739 AF352790 JX913740 JX913741 JX913742 AF260826 AY463155 EU494374 EU494360 EU494378 AF164585 EU494335 X84412 FJ447340 EU494350 EU494352 X84411 AF164584 X03240 HQ322500 DQ029097 JX913759 JX913753 JX913758 JX913757 EU154477 JX913761 JX913743 JN859549 DQ533708

Calliphora vicina Chrysomya albiceps Chrysomya bezziana Chrysomya megacephala strain DI212 Chrysomya megacephala strain DI219 Chrysomya putoria Chrysomya rufifacies strain DI215 Chrysomya rufifacies strain DI218 Chrysomya saffranea Cochliomyia hominivorax Dermatobia hominis Drosophila adunca voucher 105818 Drosophila ambigua Drosophila bostrycha Drosophila erecta Drosophila iri Drosophila lebanonensis Drosophila littoralis Drosophila mediostriata voucher 109394 Drosophila montana Drosophila nebulosa Drosophila orena Drosophila yakuba Exorista sorbillans Haematobia irritans irritans Hemipyrellia ligurriens Lucilia cuprina strain DI213.5 Lucilia porphyrina Lucilia sericata strain DI257 Musca domestica Pollenia rudis Protophormia terraenovae Sarcophaga impatiens Stomoxys calcitrans

3

Table 5 Multiplexed real-time PCR assay specificity and sensitivity (using 300 nM primers, 200 nM probe). CT score

Species

step. At the completion of the run the dynamic tube was turned on and the data was slope-corrected. After initial testing the threshold line was fixed at 0.01. For each sample a cycle threshold score (CT) was determined as the cycle number at which the DNA amplification curve crossed the threshold line. Following Jarrett et al. (2010), a sample was considered positive if the CT score was less than or equal to 35, suspicious if CT > 35, and negative if CT = 0 or no amplification. 2.3. Confirmation and optimization of multiplexed real-time PCR assay The IAC assay was tested on its own and then multiplexed with the C. bezziana specific assay (Jarrett et al., 2010). Specificity of

C. bezziana

Fly IAC detection

Chrysomya bezziana C. bezziana C. bezziana C. flavifrons C. incisuralis C. latifrons C. nigripes C. semimetallica C. putoria C. megacephala C. rufifacies C. saffranea C. varipes Cochliomyia hominivorax Hemipyrellia sp. Lucilia cuprina Musca domestica Sarcophagidae

17.5 14.5 16.4 no amp no amp no amp no amp no amp no amp no amp no amp no amp no amp no amp no amp no amp no amp no amp

19.4 20.8 20.7 22.2 19.4 18.7 19.7 21.9 15.0 18.0 18.0 20.3 23.1 15.5 18.3 17.5 17.4 18.6

Low level detection of C. bezziana 1 C. bezziana + 999 C. megacephala 1 C. bezziana + 999 C. megacephala 1 C. bezziana + 999 C. megacephala 1 C. bezziana + 999 C. megacephala

25.9 25.7 22.6 21.7

16.8 16.5 17.0 16.5

Known positive field samples ex Malaysia 4 C. bezziana in 276 total flies 1 C. bezziana in 1020 total flies 2 C. bezziana in 282 total flies 1 C. bezziana in 21 total flies 5 C. bezziana in 110 total flies 1 C. bezziana in 77 total flies

26.9 25.2 19.0 18.0 16.8 28.6

23.8 15.3 16.2 14.2 18.5 20.0

Known negative field samples ex Torres Strait 150 mixed species flies 150 mixed species flies 200 mixed species flies

no amp no amp no amp

14.7 14.0 12.1

the multiplexed assay was measured by testing DNA from 16 fly species, both individually and pooled, spanning 6 genera (Table 1). Sensitivity of the multiplexed assay was determined by testing DNA from C. bezziana, 16 non-target species and 4 samples containing 1 C. bezziana and 999 C. megacephala. Known C. bezziana-positive and C. bezziana-negative field samples were screened to further test the assay. Finally probe concentrations (50–800 nM) and primer concentrations (75–525 nM) were optimized for the assay. Positive (C. bezziana DNA) and negative (using milli-Q water instead of DNA) controls were included with each run.

Table 3 Fly internal amplification control (IAC) real-time PCR assay conserved primer and probe sequences used to target the mitochondrial DNA ribosomal 16S gene. Name

Sequence 5 -3

Position in mtDNA 16S

%GC

Annealing temp (◦ C)

FLY16SF FLY16SR FLY16S VIC PROBE

TAAATTTATTGCACTAATCTGCCAA TTAAATTCTTACATGATCTGAGTTC TTAAAGATAGAAACCAACCTGGCTTAC

65 239 180

28 28 37

59 59 69

Table 4 Cycle threshold (CT) scores for C. bezziana assay and fly internal amplification control (IAC) alone and multiplexed (using 300 nM primers, 200 nM probe). DNA

Chrysomya bezziana Mixture of 15 fly species (no C. bezziana) 1 C. bezziana in 1020 total mixed flies

Ct score C. bezziana detection in a C. bezziana assay alone

C. bezziana detection in a mulitplexed assay

Fly IAC detection in fly IAC assay alone

Fly IAC detection in mulitplexed assay

17.1 no amp 26.0

16.6 no amp 27.0

17.5 13.3 19.6

18.5 14.2 18.9

Please cite this article in press as: Morgan, J.A.T., Urech, R., An improved real-time PCR assay for the detection of Old World screwworm flies. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.02.015

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Table 6 Cycle threshold (CT) scores for probe concentration optimization (using 300 nM primers) tested against pure C. bezziana DNA. Italicized cells show the best CT scores within each assay (within +1 ◦ C of lowest CT score) with the optimal probe concentration and results across all assays in large bold font. C. bezziana assay probe concentration (BezFAM) (nM)

50 100 200 400 800

Fly IAC assay probe concentration (FLY16SVIC) (nM)

50 100 200 400 800

CT score

C. bezziana detection in C. bezziana assay alone

C. bezziana detection in mulitplexed assay

Fly IAC detection in IAC assay alone

Fly IAC detection in mulitplexed assay

16.1 16.9 17.1 17.4 18.3

17.9 16.6 17.3 17.9 no amp

19.0 18.3 17.4 17.4 19.5

20.0 18.6 20.1 21.1 no amp

3. Results Comparing the C. bezziana and fly IAC assays alone and multiplexed (Table 4) resulted in consistent detection of C. bezziana and fly DNA with congruent CT scores (±1) for each assay, whether multiplexed or not. The multiplexed assay successfully amplified DNA from all of the fly species tested with no interference detected in the FAM channel (no evidence of a false C. bezziana signal) (Table 5). The fly real-time PCR assay amplified DNA in all samples tested and negative PCR controls were negative. Sensitivity for low-level C. bezziana detection (1/1000) was confirmed with four independent laboratory spiked samples. Known C. bezziana-positive field samples from Malaysia were re-confirmed as positive and similarly known C. bezziana-negative field samples from the Torres Strait were re-confirmed as negative (Table 5). Probe optimization reactions identified the optimal C. bezziana and IAC probe concentrations for the multiplexed assay to be 100 nM (Table 6). The C. bezziana-specific assay was optimized at a 300 nM primer concentration by Jarrett et al. (2010). For this reason the C. bezziana assay primers were predominantly left at this concentration and the fly IAC primer concentrations were optimized. The lowest IAC primer concentration at which all fly templates amplified was 225 nM (Table 7). Across all of the DNA templates, increasing the IAC primer concentration largely improved (reduced) the fly CT scores. For the mixed fly templates the IAC assay showed a marked improvement in fly detection when the IAC primer concentration was increased from 225 to 300 nM. This IAC improvement corresponded to a slight drop in OWS fly sensitivity. The drop in OWS fly sensitivity was more apparent in the pure C. bezziana template. For the pure C. bezziana template, varying IAC primer concentration had little effect on OWS fly sensitivity (C. bezziana CT scores largely within ±1). However, for low-level C. bezziana detection (1/1000 flies) increasing the IAC primer concentration resulted in decreased OWS fly sensitivity. This was somewhat, but not completely, recoverable by increasing C. bezziana primer concentration to 525 nM (Table 7). 4. Discussion Recently a fluorescence-based real-time PCR assay was developed to detect C. bezziana in bulk fly trap catches (Jarrett et al., 2010). The assay exhibits good sensitivity, broad dynamic range and is capable of detecting one C. bezziana in a sample of 1000 non-target flies. An IAC assay for fly DNA has been developed to be multiplexed with the C. bezziana-specific real-time PCR assay developed by Jarrett et al. (2010). The use of two fluorogenic TaqMan® MGB probes labelled with distinct reporter dyes (FAM and VIC) allowed the simultaneous detection of the C. bezziana target and the fly IAC genes in a duplex reaction. Unlike an independent positive control, a multiplexed reaction provides an indicator

of the performance of the DNA extraction and amplification reaction in the sample tube. The multiplexed assay is also cheaper in labour and reagent costs than an independent positive control and removes tube-to-tube variability. An IAC provides an indirect measure of the integrity of the target sequence (Lion, 2001). Variable rates of degradation of single copy genes could produce false negative results. By targeting multi-copy genes for both the IAC and the C. bezziana-specific assays, the probability of detecting fly DNA is maximised even if the samples are partially degraded prior to extraction. The fly IAC assay targets the multi-copy mitochondrial DNA 16S gene and successfully amplifies DNA from 6 genera of flies commonly caught in Australian fly traps. Multiplexing of PCR reactions can lead to a decrease in assay sensitivity due to competition with the target template (Lion, 2001; Nolte, 2004). Probe and primer concentrations of the IAC were optimized to limit the competition of the C. bezziana-specific assay and the fly IAC reaction for reagents. The optimal probe concentration for the multiplexed C. bezziana and IAC assay was 100 nM. Amplification of the IAC was inhibited at the highest probe concentration (800 nM) possibly due to the probe dye interacting with sample constituents and compromising fluorescence readings (Reynisson et al., 2006). DNA is exponentially replicated in the polymerase chain reaction but reagents are limiting and cause sigmoidal amplification curves to flatten off. In a multiplexed reaction there may be competition for reagents, particularly when target DNA concentrations are heavily skewed. For this reason the optimal multiplexed C. bezziana and IAC assay should use the lowest IAC primer concentration to maximise OWS fly detection, without compromising fly detection in the IAC. Although IAC primer concentration did not compromise OWS fly detection in the pure C. bezziana samples, sensitivity was reduced in the mixed fly samples. This result reflects the changing ratio of C. bezziana to fly DNA. The mixed fly samples start with 1000-fold more fly-DNA templates than OWS fly-DNA templates and they are competing for the same PCR reagents. The optimal IAC primer concentration for OWS fly detection in the multiplexed assay is between 225 and 300 nM. For routine screening of large trap samples the recommended primer concentration for both the C. bezziana and IAC assays is 300 nM. This concentration significantly increases the sensitivity of the IAC with only a slight loss to OWS fly sensitivity. Chrysomya bezziana-positive and -negative field samples were confirmed using the multiplexed assay and fly DNA was detected in all samples. Although real-time PCR provides a quantitative measure of DNA content, scores for the OWS fly-specific and IAC assays will not necessarily scale due to differences in extraction volume associated with initial trap size, a DNA saturation effect on the Qiagen DNA extraction columns, and amount of sample degradation prior to extraction. Including the IAC in the OWS fly-detection assay provides greater confidence that a sample returning a negative assay result is truly free of OWS flies. A robust and reliable assay for early OWS fly

Please cite this article in press as: Morgan, J.A.T., Urech, R., An improved real-time PCR assay for the detection of Old World screwworm flies. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.02.015

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Table 7 Cycle threshold (CT) scores for primer concentration optimization (using 100 nM probes) and mixed C. bezziana DNA templates. Italicized cells show the best CT scores within each template concentration (within +1 ◦ C of lowest CT score) with the optimal primer concentration and results (in some instances more than one concentration) across all assays in large bold font. C. bezziana assay primer concentration (RTBezF1 & RTBezR1) (nM)

Fly IAC assay primer concentration (FLY16SF & FLY16SR) (nM)

CT score

C. bezziana detection in mulitplexed assay

Fly IAC detection in mulitplexed assay

Pure C. bezziana DNA 300 300 300 300 300 300 300 375 450 525

75 150 225 300 375 450 525 375 450 525

17.4 16.7 16.9 18.2 17.0 16.4 17.3 16.8 16.9 16.6

no amp no amp 20.0 19.7 18.9 18.5 16.5 19.0 19.0 17.8

1 C. bezziana + 999 C. megacephala DNA 300 300 300 300 300 300 300 375 450 525

75 150 225 300 375 450 525 375 450 525

19.7 19.3 19.9 20.7 21.6 23.4 22.9 20.6 22.1 21.0

no amp no amp 18.6 13.8 14.9 14.3 14.4 15.9 14.7 13.3

1 C. bezziana + 20 mixed fly species (Field sample) 300 300 300 300

75 150 225 300

17.0 17.2 17.2 18.0

no amp 19.7 16.5 14.2

detection will be essential in the event of an incursion. Early detection will assist in controlling the spread of OWS fly in Australia, thereby reducing the serious economic damage and animal welfare impacts on livestock and wildlife exposed to C. bezziana. The technique is not limited to Australia; it could be applied as a biosecurity tool anywhere there is a risk of OWS fly incursion. Acknowledgements The authors would like to thank Drs Peter Green, Philip Spradbery, James Wallman, Jeff Wells, Steven Skoda and Sri Muharsini for providing the flies used in this study. Additional thanks to Drs Sri Muharsini and April Wardhana for providing colony OWS flies for assay development and testing and to Dr Peter James for editorial feedback. References Anaman, K.A., Atzeni, M.G., Mayer, D.G., Stuart, M.A., Butler, D.G., Glanville, R.J., Walthall, J.C., Douglas, I.C., 1993. Economic Assessment of the Expected Producer Losses and Control Strategies of a Screwworm Fly Invasion of Australia: Screwworm Fly Bio-Economic Decision Aid Modelling Project. Project Report. Department of Primary Industries, Brisbane, Australia, pp. 108. Anaman, K.A., Atzeni, M.G., Mayer, D.G., Walthall, J.C., 1994. Economic-assessment of preparedness strategies to prevent the introduction or the permanent establishment of screwworm fly in Australia. Prev. Vet. Med. 20, 99–111. Animal Health Australia, 2012. Request for tender: The risks of entry of screw-worm fly into Australia and surveillance requirements. . (accessed 9 Sep 2013). AUSVETPLAN, 2007. Australian Veterinary Emergency Plan, Disease Strategy, Screwworm Fly Technical Review Group. Animal Health Australia, Deakin, ACT, Australia. Guo, Y., Cai, J., Chang, Y., Li, X., Liu, Q., Wang, X., Wang, X., Zhong, M., Wen, J., Wang, J., 2011. Identification of forensically important Sarcophagid flies (Diptera:

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Please cite this article in press as: Morgan, J.A.T., Urech, R., An improved real-time PCR assay for the detection of Old World screwworm flies. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.02.015

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Please cite this article in press as: Morgan, J.A.T., Urech, R., An improved real-time PCR assay for the detection of Old World screwworm flies. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.02.015

An improved real-time PCR assay for the detection of Old World screwworm flies.

The Old World screwworm (OWS) fly, Chrysomya bezziana, is a serious pest of livestock, wildlife and humans in tropical Africa, parts of the Middle Eas...
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