Development of a TaqMan real-time RT-PCR assay for detection of covert mortality nodavirus (CMNV) in penaeid shrimp

    Development of a TaqMan real-time RT-PCR assay for detection of covert mortality nodavirus (CMNV) in penaeid shrimp Chettupon Pooljun, Sataporn Direkbusarakom, Piyapong Chotipuntu, Ikuo Hirono, Suwit Wuthisuthimethavee PII: DOI: Reference:

S0044-8486(16)30347-7 doi: 10.1016/j.aquaculture.2016.06.044 AQUA 632219

To appear in:

Aquaculture

Received date: Revised date: Accepted date:

11 April 2016 29 June 2016 30 June 2016

Please cite this article as: Pooljun, Chettupon, Direkbusarakom, Sataporn, Chotipuntu, Piyapong, Hirono, Ikuo, Wuthisuthimethavee, Suwit, Development of a TaqMan realtime RT-PCR assay for detection of covert mortality nodavirus (CMNV) in penaeid shrimp, Aquaculture (2016), doi: 10.1016/j.aquaculture.2016.06.044

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ACCEPTED MANUSCRIPT Development of a TaqMan real-time RT-PCR assay for detection

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of covert mortality nodavirus (CMNV) in penaeid shrimp

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Chettupon Pooljun1, Sataporn Direkbusarakom1, Piyapong Chotipuntu1, Ikuo Hirono2 and

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Center of Excellence for Shrimp, School of Agricultural Technology, Walailak University,

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Nakhon Si Thammarat 80160, THAILAND 2

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Suwit Wuthisuthimethavee1*

Laboratory of Genome Science, Department of Marine Biosciences, Faculty of Marine

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Science, Tokyo University of Marine Science and Technology, Tokyo 108-8477, JAPAN

*Correspondence should be addressed to:

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Asst. Prof. Suwit Wuthisuthimethavee Center of Excellence for Shrimp, School of Agricultural Technology, Walailak University,

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Nakhon Si Thammarat 80160, THAILAND. E-mail: [email protected]

ACCEPTED MANUSCRIPT ABSTRACT

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Covert mortality nodavirus (CMNV) is an RNA virus that infects shrimp and is responsible for economic losses in the shrimp aquaculture industry in several countries of

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Southeast Asia and China. Detection of the disease at an early stage can enhance proper farm management. However, the recent disease monitoring technique of confining nested reversetranscription PCR (RT-PCR) is time consuming and does not determine the viral load in

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infected shrimp. This study develops a more efficient technique for the detection of CMNV in

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infected shrimp using TaqMan probe-based real-time RT-PCR. The procedure comprises the isolation of CMNV from infected shrimp and the isolation of the messenger RNA to construct

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a complementary DNA template for PCR. Viral detection viability was tested using both nested RT-PCR and real-time RT-PCR. The results showed that real-time RT-PCR is highly

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sensitive in terms of minimal viral copy numbers compared to nested RT-PCR results. Realtime RT-PCR has viral detection capability as small as 1 copy number of the CMNV plasmid

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while conventional RT-PCR and nested RT-PCR recognize minimal detectable numbers of

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10,000 and 100 viral copies in shrimp sample, respectively. The results from real-time RTPCR showed the viral load in shrimp samples varied from 4.27 to 6.53×106 copies number, while nested RT-PCR recognize minimal detectable numbers of 281 viral copies. The realtime RT-PCR technique developed in this study is advantageous because it is less time consuming compared to the nested RT-PCR technique, works well in viral load analysis and can exclude cross-reaction with other shrimp RNA viruses when tested against TSV, YHV, and IMNV. Statement of Relevance This study developed methods for CMNV detection in penaeid shrimp. Keywords: covert mortality nodavirus (CMNV); TaqMan probe; real-time RT-PCR; shrimp

ACCEPTED MANUSCRIPT 1. INTRODUCTION In the last decade, epidemic diseases have devastated the shrimp industry, especially

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in Asian and Southeast Asian countries that include India, China, Malaysia, Indonesia, the Philippines, Vietnam, and Thailand (Moffitt and Cajas-Cano, 2014). This has caused a

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decrease in average global production of farmed shrimp from 4 million tons in 2011 to 3.3 million tons in 2013. Production recovered slightly and increased to 3.7 million tons in 2015 because the outbreak of diseases was partially overcome but did not reach full recovery levels

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(Chamberlian, 2014).

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Covert mortality nodavirus (CMNV), a new nodaviridae virus-borne disease affecting crustaceans, was first detected in China in 2009 (Zhang et al., 2014). CMNV is a positive

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single-stranded RNA virus that is spherical and non-enveloped with a diameter of approximately 24.9±1.8 nm (Zhang et al., 2014). The disease causes economic losses in

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hatcheries and farms due to high mortality rates of up to 80% commonly found within 60-80 days post-stocking. Clinical signs of CMNV infection in shrimp are whitish muscle necrosis,

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coloration in the abdominal muscle, hepatopancreatic atrophy and necrosis, empty stomach

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and gut, soft shell, and slow growth. These clinical signs are comparable to those of shrimp infected by Macrobrachium rosenbergii nodavirus (MrNV) and Penaeus vannamei nodavirus (PvNV) of the nodaviridae family found in Macrobrachium rosenbergii and Penaeus vannamei, respectively (Arcier et al., 1999; Ravi et al., 2009; Tang et al., 2007; Yoganandhan et al., 2006). Infected shrimp are commonly found in deep water on the bottom of their environment rather than swimming on the surface or in shallow water. The first CMNV detection was reported in 2014. The detection of CMNV in shrimp is done using nested RT-PCR, where RNA is extracted from purified virus samples and infected shrimp. The cDNA is further synthesized using specific primer for nested PCR (Zhang et al., 2014). The nested RT-PCR is time consuming and less sensitive compared to real-time PCR

ACCEPTED MANUSCRIPT and real-time RT-PCR. Both techniques are powerful methods that can be practically used to detect and quantify both bacterial and viral shrimp pathogens such as Vibrio

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parahaemolyticus, Salmonella spp., infectious hypodermal and hematopoietic virus (IHHNV), Monodon baculovirus (MBV), white spot syndrome virus (WSSV), yellow head

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virus (YHV), and Taura syndrome virus (TSV) (Durand and Lightner, 2002; Tang and Lightner, 2001; Yan et al., 2009). Currently, the specific real-time reverse transcription loopmediated isothemal amplification (RT-LAMP) was developed to detect CMNV in

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Litopenaeus vannamei shrimp (Zhang et al., 2015). This assay is rapid and higher sensitive

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than nested RT-PCR but requires complicated processes for optimizing the reactions and restricts to the availability of reagents, specific primer and instruments. However, to quantify

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the target virus, the real-time PCR technique requires a fluorescent-oligonucletide probe specific for viral species which is not available for CMNV. In this study, we thus aimed to

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develop a TaqMan probe-based real-time RT-PCR technique in order to establish a more

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efficient technique for detection of CMNV infection in shrimp.

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2. MATERIALS AND METHODS 2.1 Sample preparation A total of sixty-nine shrimp that showed clinical signs of CMNV infections including death shrimp, and shrimp of whitish muscle necrosis, abdominal muscle coloration, hepatopancreatic atrophy and necrosis were collected from shrimp farms in four southern provinces of Thailand. The samples were tested to verify that they had not been infected by other virus and bacteria including WSSV, IHHNV, TSV, YHV, infectious mionecrosis virus (IMNV) and Vibrio parahaemolyticus. Shrimp samples infected with other RNA viruses (i.e., TSV, YHV, or IMNV) were also utilized to determine the cross reaction of other RNA virus with CMNV.

ACCEPTED MANUSCRIPT 2.2 Total RNA isolation Total RNA of the samples was extracted using the TRIsure kit (Bioline, Tuanton, MA,

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USA) following the protocol for RNA isolation. Briefly, total RNA was extracted from 100 mg of shrimp tissue using 1 ml of TRIsure and homogenization. After incubation for 5 min at

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room temperature, 200 µl of chloroform was added and the solution was vigorously mixed. The tube was incubated for 3 min before centrifugation at 12,000 ×g for 15 min at 4C. The aqueous phase was transferred to a fresh tube, 500 µl of 100% cold isopropyl alcohol was

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added and gentle mixed. After incubation for 10 min at room temperature, the tube was

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centrifuged at 12,000 ×g for 10 min at 4 C and the pellet was rinsed with 75% (v/v) ethanol, air dried, and dissolved in 50 µl of RNase-free water. RNA concentration and quality were

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assessed by spectrophotometer at wavelengths of 260 and 280 nm (NanoDrop 2000c

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Spectrophotometer, Thermo Fisher Scientific, Wilmington, DE, USA).

2.3 Reverse-transcription PCR

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The iScript Select cDNA Synthesis kit protocol (BIO-RAD, Hercules, CA, USA) was

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used to synthesize cDNA in a total reaction volume of 10 µl. The reaction mixture of the iScript selected cDNA synthesis kit was prepared from 1 µl of total RNA (1 µg/µl), 2 µl of 5x iScript select reaction mix, 0.25 µl of CMNV-R primer (10µM), 1 µl of GSP enhancer solution, 0.5 µl of iScript reverse transcriptase, and 5.25 µl of nuclease-free water. To perform RT-PCR, the RNA sample was activated at 42C for 30 min and then incubated at 85C for 5 min to inactivate the reverse transcriptase. The cDNA product was further amplified by real-time PCR for CMNV detection.

2.4 CMNV positive sample

ACCEPTED MANUSCRIPT The nested RT-PCR which was developed by Zhang and colleagues (Zhang et al., 2014) was used to determine the positive CMNV in shrimp samples. Briefly, the cDNA was

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synthesized and amplified using specific primer for nested PCR. The products from nested PCR showed a 618 bp amplicon for the first step and a 165 bp amplicon for the second step.

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The products were visually observed in 1% agarose gel electrophoresis.

2.5 Selection of PCR primers and TaqMan probe for CMNV

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A pair of RT-PCR primers and a TaqMan probe (Table. 1) were designed from the

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RNA-dependent RNA polymerase (RdRp) gene of CMNV from which 130-bp amplicon (981-1110 in GenBank KM112247.1) was obtained. The CMNV probe was synthesized and labeled with 5-carboxyfluorescein (FAM) dye at the 5’ end and carboxytetramethylrhodamine

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(TAMRA) at the 3’ end (5’ FAM/3’ TAMRA).

2.6 Control CMNV plasmid and standards quantification

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A 10-fold dilution of purified CMNV plasmid (pCMNV) was prepared in order to

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construct the standard curve. The concentration of plasmid ranged from 108 to1 copy per reaction. The plasmid was used as PCR positive control template and for determination of sensitivity of the real-time PCR. For the pCMNV, the CMNV plasmid (pMD19-T) containing fragment from the RNAdependent RNA polymerase gene of CMNV was obtained from the Chinese Academy of Fishery Sciences (CAFS), China.

2.7 Real-time RT-PCR assay and sequencing analysis To evaluate the real-time RT-PCR, 69 RNA shrimp samples that had been tested by nested RT-PCR were used for detection of CMNV by real-time RT-PCR. The real-time RT-

ACCEPTED MANUSCRIPT PCR was performed on ABI 7300 Real time PCR System (Applied Biosystem). The reaction mixture contained 1µl of cDNA, 2 µl of master mix (5x HOT FIREpol Probe qPCR Mix Plus

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(ROXX) (Solis Biodyne, Tartu, Estonia), 0.25 µl of each primer at a concentration of 10 µM (Table 1), and 6.5 µl of water, for a final reaction volume of 10 µl. The shrimp samples were

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evaluated in triplicate for each mixture. A 10- fold dilution of pCMNV was used as a standard in the same condition. The standard curves were evaluated in duplicate for each mixture tested. The cycling consisted of 15 min at 95C followed by 40 cycles of 95C for 30

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s and 60C for 1 min. The data were analyzed using the 7300 Real Time PCR software. To

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confirm the TaqMan assay results, the real-time PCR products were subjected to electrophoresis on 1.5% agarose gel and photographed.

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For sequencing, the CMNV-positive RNA sample from shrimp was amplified and purified using the GenepHlow Gel/PCR kit (Geneaid Biotech, New Taipei City, Taiwan) then

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sequenced with an automated DNA sequencer, the ABI Prism 3730xl genetic analyzer (Applied Biosystems) at Bio Basic Inc. in Canada. A basic local alignment search tool

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database.

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(BLAST) search was used to align the sequence of the CMNV PCR product with the NCBI

2.8 Comparative analysis of real-time RT-PCR, conventional RT-PCR, and nested RTPCR The cDNA product obtained from CMNV positive shrimp by real-time RT-PCR (4.63×106 copies number) was 10-fold serially diluted to determine the effect of different concentrations of template cDNA of conventional RT-PCR. The PCR mixture reaction was prepared from 1µl of cDNA, 2 µl of 5x FIREPol Master Mix (Solis Biodyne, Tartu, Estonia), 0.25 µl of each primer at a concentration 10 µM (CMNV-F and CMNV-R (Table 2), and 6.5 µl of water, for a final reaction volume of 10 µl. The cycling conditions were 95 C for 3 min,

ACCEPTED MANUSCRIPT followed by 35 cycles at 95C for 30 s, 60C for 30 s, and 72C for 30 s and a final extension step of 72C for 5 min. The products were observed in 1% agarose gel electrophoresis. The

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expected target amplicon size was 130 bp. Purified pCMNV was used as a positive control.

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3. RESULTS 3.1 Positive CMNV in shrimp samples

The nested RT-PCR results for CMNV detection indicated that 21 of 69 samples were

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CMNV positive. The CMNV products were resolved by 1% agarose gel electrophoresis. The

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amplification of the template produced a band of the expected size of 618 bp for the first step

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(Fig.1a) and a 165 bp for the second step (Fig.1b).

3.2 Sensitivity of real-time RT-PCR

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To determine the sensitivity of real-time RT-PCR, 10-fold serially diluted CMNV plasmid which containing the target sequence was used as a template for preparing the

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standard. The TaqMan real-time RT-PCR gave linear curves for 108 to 1 copies of pCMNV

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(Fig. 2a). The sensitivity per reaction of the assay was as high as 1 copy detection of pCMNV with correlation coefficient (R2) of 0.9915 and a slope of 3.002 (Fig. 2b). These values were used to calculate the number of CMNV RNA copies in shrimp samples.

3.3. Detection samples and specificity of real-time RT-PCR To detect CMNV in the shrimp samples by real-time PCR, a 10-fold serial dilution of pCMNV was used as the standard. The sensitivity of the assay was determined with correlation coefficient (R2) of 0.9960 and a slope of 3.3064 (Fig. 3a-b). 26 of the 69 samples were found to be CMNV positive using real-time RT-PCR (Table 2). This includes all samples that are found positive for CMNV tested by nested RT-PCR. The amplification plot

ACCEPTED MANUSCRIPT of the CMNV infected shrimps showed a range of 106 to 100 copies (Fig.3c) and the viral loads of the positive clinical samples varied within the range 4.27 to 6.53×106 copy number

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per 1 µl of total RNA (Table 2). Moreover, the real-time RT-PCR assay showed that 5 samples from the 48 CMNV-negative samples determined by nested RT-PCR gave positive

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results. The diagnostic sensitivity (DSe) and diagnostic specificity (DSp) for real-time RTPCR assay compared with the nested RT-PCR were 100% and 89.6%, respectively. The specificity (cross-amplification) of real-time RT-PCR in Figure 3d shows that no

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amplification was observed using total genomic RNA from the shrimp infected with TSV,

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YHV, IMNV, and no template control (NTC) (Fig. 3d), which was confirmed by electrophoresis (Fig. 3e). This also indicates that the amplifications of the primer sets are

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specific to the target virus and clearly amplified.

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3.4. Viral sequencing

A total of 100 bp from a total 130 bp of CMNV PCR product was sequenced. The

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results showed 99% of the nucleotide from CMNV PCR product was identical to the

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nucleotide sequences of the CMNV isolate Hebei-20120627 RNA-dependent RNA polymerase (RdRp) gene (GenBank accession number KM112247.1). This indicates that the PCR product amplified using this primer (Table 1) is CMNV.

3.5. The sensitivity of conventional RT-PCR and nested RT-PCR The cDNA synthesized from the CMNV positive sample (6.53× 106 copies) by realtime RT-PCR was used to set up 10-fold serial dilutions used as templates for amplification. The real-time RT-PCR showed high sensitivity to CMNV detection at a concentration of 1 copy, whereas conventional RT-PCR presented the best sensitivity of 104 cDNA copies (Fig. 4a), and the nested RT-PCR detected cDNA beyond 102 copies (Fig. 4b).

ACCEPTED MANUSCRIPT 4. DISCUSSION This study applied real-time PCR with a TaqMan probe to quantify CMNV in infected

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shrimp. The results indicate that this method is highly specific for CMNV detection comparing with conventional RT-PCR and nested RT-PCR. In this report, among the 69

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shrimp samples, 21 (30.4%), and 26 (37.7%) were positive by nested RT-PCR, and real-time RT-PCR, respectively. All of the positive samples by nested RT-PCR were also positive by real-time RT-PCR. The detection limits of this real-time RT-PCR were 1 copy of CMNV

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plasmid and 4.27 copies of clinical shrimp sample, the correlation coefficient was high (r2 =

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0.9960). These results are similar to those reported for RT-LAMP assays for CMNV detection, the detection limit of the RT-LAMP assay was 27 copies of the target plasmid

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(Zhang, et al., 2015). This study showed that the relative sensitivity of the CMNV real-time RT-PCR assay was slightly higher than that of the RT-LAMP method. Furthermore, real-time

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RT-PCR exhibited no cross-reaction with SPF shrimp, TSV, YHV and IMNV-infected penaeid shrimp, indicating that real-time RT-PCR is specific to CMNV. The primers and

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shrimp.

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probe designed in our study were sensitive to CMNV isolated from CMNV infected penaeid

The real-time RT-PCR is a more rapid technique than nested RT-PCR for CMNV detection. It is capable of detection small amount of cDNA templates and requires only 1.30 h while the nested PCR usually takes 2 h for steps 1 and 2 of amplification and 1 h for electrophoresis. Moreover, it is more sensitive than conventional RT-PCR and nested RTPCR, which are able to detect 104 and 102 copies, respectively. It requires less than 10 copies of cDNA and is 10 times and 1000 times more sensitive than nested RT-PCR and conventional RT-PCR, respectively in terms of detectable copy number. In addition, nested RT-PCR has a higher risk of inaccurate results due to contamination (Porter-Jordan et al., 1990).

ACCEPTED MANUSCRIPT In the last decade, several TaqMan-based real-time PCR methods have been developed for detection of disease in aquaculture infected by DNA and RNA viruses (Durand

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and Lightner, 2002; Liu et al., 2013; Schikorski et al., 2013; Tang et al., 2004; Yan et al., 2010). In the case of penaeid shrimp infected by an RNA virus, real-time RT-PCR was used

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to determine the viral copy number for shrimp infected with TSV. The technique can detect at least 100 copies of purified TSV RNA (Nunan et al., 2004) and 2x103 copies of TSV RNA copies (Cao et al., 2010). For IMNV, the detection limit of real-time RT-PCR was 10 copies,

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which is 100-fold more sensitive than nested RT-PCR (Andrade et al., 2007). For YHV, the

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detection limit of real-time RT-PCR was 102 copies of YHV, which is more sensitive than conventional RT-PCR (Ma et al., 2008).

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Real-time RT-PCR has been well recognized as offering several advantages over nested PCR. In addition to allowing quantification of the viral load, it reduces the risk of

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amplicon contamination and the time required for response. It also offers a safer laboratory protocol as ethidium bromide which is a carcinogen is not used. The real-time PCR technique

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exhibited sufficient sensitivity, specificity, and quantitative detection capability to be used as

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a powerful tool for microbiological examination of shrimp diseases (Leal et al., 2013)

CONCLUSION

The real-time RT-PCR utilizing a TaqMan probe developed in this study is a feasible technique for detection of CMNV in penaeid shrimp. It is highly sensitive method and less time consuming. This technique contributes to innovation regarding effective determination of shrimp viral diseases comparing with conventional nested RT-PCR.

ACKNOWLEDGMENT

ACCEPTED MANUSCRIPT The authors would like to thank the Center of Excellence for Shrimp at Walailak University for providing experimental facilities. The authors also wish to thank Prof. Jie

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Huang and Dr. Qing-Li Zhang from the Chinese Academy of Fishery Sciences (CAFS) for

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providing the CMNV plasmid.

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FIGURE LEGENDS Figure 1 Nested RT-PCR detection of CMNV in shrimp that showed the clinical signs of CMNV: (A) the first step PCR products from nested PCR showed a 618 bp; (B) the second

ACCEPTED MANUSCRIPT step PCR products showed a 165 bp where M in both (A) and (B) is a ladder (100 bp DNA ladder, GeneDirex), lane 1-4 are shrimp samples, lane P is a CMNV plasmid and lane N is

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NTC.

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Figure 2 Sensitivity of the pCMNV real-time PCR test: (A) log scale amplification plots (Rn: fluorescence signal) and (B) CMNV standard curve produced using 10-fold serial dilutions of

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pCMNV as a standard template and values of slope, intercept, and correlation (R2).

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Figure 3 Sensitivity and specificity of the real-time RT-PCR assay for detection of CMNV RNA from shrimp samples: (A) log scale amplification plots of a 10-fold serially dilution of pCMNV from 102 (1) to 108 (7) copies/ µl; (B) standard curve and values of slope, intercept,

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and correlation (R2); (C) log scale amplification plots of the CMNV-positive shrimp samples

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where P is pCMNV and 1-6 are shrimp samples; (D) log scale amplification plots of the CMNV-positive shrimp samples where lane P is pCMNV, lane 3 is CMNV-positive shrimp

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samples (1.43×103 CMNV copy number) and lane 7-11 are IMNV, TSV, YHV, SPF, and

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NTC, respectively; (E) Electrophoresis of real-time RT-PCR products where M is a ladder (100 bp DNA ladder, Solis Biodyne, Tartu, Estonia), lane P is pCMNV, lanes 1-6 are shrimp samples, lane 7-9 are other RNA viruses (IMNV, TSV and YHV, respectively), lane 10 is SPF shrimp and lane 11 is NTC.

Figure 4 Agrose gel electrophoresis: (A) Conventional RT-PCR products using CMNV-F and CMNV-R primers where M is a ladder (100 bp DNA ladder, Solis Biodyne, Tartu, Estonia), lanes 1-7 are 10-fold diluted cDNA from CMNV infected shrimp (100 to 106 copies, respectively), lane 8 is a pCMNV and lane 9 is a NTC; (B) 2nd PCR product using Zhang et al.’s (2014) primers where lane M is a ladder (100 bp DNA ladder, Solis Biodyne, Tartu,

ACCEPTED MANUSCRIPT Estonia), lanes 1-7 are 10-fold diluted cDNA from CMNV infected shrimp (100 to 106 copies,

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respectively), lane 8 is a pCMNV and lane 9 is a NTC.

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Figure 3

ED

MA

NU

SC RI

PT

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AC

CE

PT

Figure 4

ACCEPTED MANUSCRIPT Table 1 Primers used in the study. Nucleotide sequence

CMNV-F

5’-ACC TCC GCA ATC TGA TTG-3’

CMNV-R

5’-GGG TCT ACT TTC GTT GGA-3’

CMNV-TaqMan

5’-CGC TAC CAC TGTC GGC TTG T-3’

AC

CE

PT

ED

MA

NU

SC RI

PT

Primers/Probe

ACCEPTED MANUSCRIPT Table 2 Summary of the CMNV positive results using real-time RT-PCR compared with nested RT-PCR of shrimp samples.

a

CE

Means of triplicate copy number (-) Detected as negative by nested RT-PCR.

AC

SC RI NU

MA

+ + + + + + + + + + + + + + + + + + + + +

PT

Nested RT-PCR

ED

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 16 19 20 21 22 23 24 25 26

Real-time RT-PCRa (CMNV copies/µl RNA) 4.63 × 106 1.13 × 105 1.43 × 103 1.11 × 103 2.81 × 102 4.27 2.63 × 105 2.25 × 104 6.81 × 105 9.65 × 105 13.22 2.13 × 104 5.41 × 102 9.32 × 103 37.94 3.83 × 104 42.76 2.62 × 104 57.17 4.35 × 106 8.88 × 104 1.49 × 104 7.84 × 105 9.71 × 103 6.53 × 106 1.09 × 104

PT

Shrimp no.

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AC

CE

PT

ED

MA

NU

SC RI

PT

Highlights 1. Real-time RT-PCR is highly sensitive in terms of minimal viral copy numbers. 2. Real-time RT-PCR technique is less time consuming and can exclude cross-reaction with other shrimp RNA viruses. 3. The detection threshold of the assay is 1 copy of CMNV plasmid and 4.27 viral copies of shrimp sample.