Development and validation of a TaqMan RT-qPCR for the detection of convert mortality nodavirus (CMNV)

Accepted Manuscript Title: Development and validation of a TaqMan RT-qPCR for the detection of convert mortality nodavirus (CMNV) Authors: Xiao-Ping Li, Xiao-Yuan Wan, Ting-Ting Xu, Jie Huang, Qing-Li Zhang PII: DOI: Reference:

S0166-0934(18)30023-5 https://doi.org/10.1016/j.jviromet.2018.10.001 VIRMET 13542

To appear in:

Journal of Virological Methods

Received date: Revised date: Accepted date:

18-1-2018 5-10-2018 5-10-2018

Please cite this article as: Li X-Ping, Wan X-Yuan, Xu T-Ting, Huang J, Zhang Q-Li, Development and validation of a TaqMan RT-qPCR for the detection of convert mortality nodavirus (CMNV), Journal of Virological Methods (2018), https://doi.org/10.1016/j.jviromet.2018.10.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Development and validation of a TaqMan RT-qPCR for the detection of convert mortality nodavirus (CMNV) Xiao-Ping Li1, 2, Xiao-Yuan Wan1, Ting-Ting Xu1, 2, Jie Huang1, Qing-Li Zhang1, 2* 1

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Function Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology; Key Laboratory of Maricultural Organism Disease Control, Ministry of Agriculture; Qingdao Key Laboratory of Mariculture Epidemiology and Biosecurity; Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences; Qingdao 266071, China; 2 National

Demonstration Center for Experimental Fisheries Science Education (Shanghai Ocean University), Shanghai 201306, China *Corresponding

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author. Tel: +86 532 85823062; Fax: +86 532 85811514 E-mail address: [email protected] (Qingli Zhang);

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Highlights

• A one-step, quantitative method for CMNV was developed.

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• The assay had a detection limit of 5.7 viral copies. • The assay was no cross-reactivity observed with six common shrimp viruses.

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• This is a useful tool for CMNV detection and quantification.

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ABSTRACT: Covert mortality nodavirus (CMNV), an emerging RNA virus, is the pathogen of viral covert mortality disease (VCMD), which has emerged as a cause of serious losses in shrimp aquaculture in China. To improve VCMD diagnosis, a one-step, real-time TaqMan probe-based reverse transcription quantitative PCR (RT-qPCR) was developed in this study. The TaqMan RT-qPCR was optimized firstly, whereby the best results were obtained with 0.2 μM of each primer, 0.2 μM probe, and 0.5 μL Enzyme Mix II. The optimal reaction program was determined as 15 min at 51ºC for reverse transcription and 5 min at 95 ºC, followed by 40 cycles of denaturation at 94 ºC for 10 s, and annealing and extension at 52.7 ºC for 30 s. The optimized assay detected as little as 9.6 pg total RNA from CMNV-infected shrimp and 5.7 copies of the target plasmid. The RT-qPCR assay for CMNV with a high correlation coefficient (r2 = 0.996) was developed basing on the standard curve generated by plotting the threshold cycle values (y) against the common logarithmic copies (log10nc as x; nc is copy number) of pMD20-CMNV. The diagnostic sensitivity and specificity of this assay versus the previously reported RT-qPCR was 96.2% and 98.0%, respectively. This method is highly specific to CMNV, as it showed no 1

cross-reactivity with other common shrimp viruses. It is anticipated that the newly developed and optimized RT-qPCR assay will be instrumental for the rapid diagnosis and quantitation of CMNV. Keywords: covert mortality nodavirus; CMNV; TaqMan; RT-qPCR

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Introduction Covert mortality disease (CMD) was initially observed in high density farming ponds of Penaeus vannamei prior to 2009 in southern China and caused substantial shrimp mortality on the affected farms (Zhang, 2004; Song & Zhuang, 2006; Xu & Ji, 2009; Gu, 2012). Cumulative mortality of P. vannamei in the CMD-affected ponds was variable but reached up to ~80-90% (Zhang, 2004; Xing, 2004; Zhang et al., 2014). A new RNA virus, covert mortality nodavirus (CMNV), was proven to be the infectious agent of CMD (Zhang et al., 2014), after which CMD was renamed viral covert mortality disease (VCMD) (Zhang et al., 2017). Epidemiological surveys revealed that CMNV was prevalent in the cultured shrimps and prawns, including P. vannamei, P. chinensis, P. japonicus, P. monodon, and Macrobrachium rosenbergii from 2013 to 2015, and 31.8% of crustaceans samples collected from the coastal provinces of China were found to be infected by CMNV (Zhang et al., 2017). Meanwhile, two research groups reported 43% and 37.7% high prevalence of CMNV in the cultured shrimp in Thailand, respectively. (Pooljun et al., 2016; Thitamadee et al., 2016). The high prevalence and wide geographical distribution of CMNV suggested a high risk for global transmission. To reduce the risk of transmission and facilitate disease management, early and accurate detection of CMNV is crucial. Currently, approaches for the detection of CMNV include a reverse transcription nested PCR (RT-nPCR; Zhang et al., 2014) and a real-time reverse transcription PCR (RT-PCR; Pooljun et al., 2016). However, the RT-nPCR is relatively time-consuming and is somewhat lacking in sensitivity. The previously reported real-time RT-PCR (Pooljun et al., 2016) was an ideal detection method for CMNV, but we found that its detection specificity was challenged by new variant strains of CMNV. For instance, it could not work well for some CMNV-positive samples determined by in situ hybridization and electron microscope analyses. The present study aimed to develop a new TaqMan-based real-time reverse transcription quantitative PCR (RT-qPCR) assay targeting the CMNV RNAdependent RNA polymerase gene for the detection and quantification of all known CMNV variant strains. Furthermore, the sensitivity, specificity, and reproducibility of CMNV detection, along with the clinical application of this assay, were evaluated. Materials and methods Collection of shrimp samples From 2016 to 2017, a total of 225 live shrimp samples including P. vannamei, P. chinensis, P. japonicus, P. monodon, and Exopalaemon carinicauda, ranging from 2~15 gram in weight, were collected from Shandong Jiangsu and Zhejiang provinces in China. The cephalothoraxes and muscles from every individual were divided into three parts and preserved respectively in RNAlater RNA Stabilization Reagent 2

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(Qiagen GmbH, Hilden, Germany), Davidson’s alcohol formalin acetic acid (AFA) solution (Lightner, 1996) or TEM fixative (2% paraformaldehyde, 2.5% glutaraldehyde, 160 mM NaCl and 4 mM CaCl2 in 200 mM PBS, pH 7.2). The samples stored in RNAlater, AFA and TEM fixative were then used for molecular, histopathological, and electron microscopic evaluation, respectively. RNA extraction and purification Total RNA was extracted from the hepatopancreas of each sample by using a commercial RNA Rapid Extraction Kit (Bio Teke, Beijing, China) following the manufacturer's instructions. The concentration and quality of the extracted RNA was measured by Nanodrop 2000 (Thermo Scientific, Waltham, MA, USA) according to the instrument instructions. Design of primers and probe Previous sequencing results have shown that for the majority of CMNV isolates the RNA-dependent RNA polymerase (RdRp) gene is conserved, with a few isolates demonstrating slightly more variability in partial regions of RdRp gene. To develop a TaqMan RT-qPCR assay specific to CMNV, a partial comparatively conserved fragment of RdRp gene was chosen as the target gene for this assay. The oligonucleotide primers and probe for the TaqMan RT-qPCR assay were designed using the Beacon Designer software version 7.0 (PREMIER Biosoft, Palo Alto, CA, USA), using the target gene sequence of the CMNV RNA-dependent RNA polymerase gene (GenBank accession number KM112247; Fig.1). The oligonucleotide primers designed in this study included the forward primer CMNV-Taq-F (5ʹ- CGA GCT AAT CCA AGC ACT TC -3ʹ) and reverse primer CMNV-Taq-R (5ʹ- ACC TGT TAG GTA CGC TAC CA -3ʹ), which yield an amplicon of 198 bp. The sequence of the TaqMan probe was 5ʹ- FAM CGC TCA CGG CTT TGG ATA CCT T TAMRA-3ʹ, which was labeled with a 6-carboxyfluorescein (FAM) reporter dye at the 5ʹ end and with a TAMRA™ quencher (TAMRA) at the 3ʹ end. The primers and probe were synthesized by SANGON (Shanghai, China) and purified by high-performance liquid chromatography (HPLC). Optimization of the CMNV TaqMan RT-qPCR Assay The CMNV TaqMan RT-PCR was performed in a BIORAD CFX96 Touch™ Real-Time PCR Detection System (BIORAD, Hercules, California, USA) using a 25 μL reaction mixture containing 12.5 μL 2× One Step RT-PCR Buffer III (Takara), varying concentrations of PrimeScript RT Enzyme Mix II (Takara), varying concentrations of primers and probe, 0.5 μL Ex Taq HS (Takara), and 20 ng total RNA of CMNV-infected shrimp or 105 copies of the target plasmid as the template. The basic reaction program included reverse transcription at 50ºC for 30 min and degeneration at 94ºC for 5 min, followed by 40 cycles of denaturation at 94ºC for 10 s, and annealing and extension at 60 ºC for 30 s. Next, the incubating temperature for reverse transcription was set as 45ºC, 46ºC, 48ºC, 50.8C, 54.5ºC, 57.5ºC, 59.1ºC, and 60ºC for optimization. Afterwards, the duration of reverse transcription was set as 10 min, 15 min, 20 min and 25 min for optimization. Next, the annealing temperature of the reaction was set as 52.0ºC, 52.7ºC, 54.0ºC, 55.9ºC, and 58.4ºC for optimization 3

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and the concentrations of probe and primers were set as 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM and 0.6 μM. Finally, the volume of PrimeScript RT Enzyme Mix II was set at 0.2 μL, 0.4 μL, 0.6 μL, 0.8 μL, and 1.0 μL for optimization. The TaqMan RT-qPCR run was performed in triplicate and no-template controls were included in each run. The optimal temperature, duration, and concentration were determined as the one with the lowest threshold cycle (Ct) and the lowest standard deviation (SD). The optimal parameters were used in the subsequent experiments. Construction of the recombinant standard plasmid The TaqMan RT-qPCR products of primers of CMNV-Taq-F and CMNV-Taq-R were subjected to sequencing for validation. Then the PCR products were purified using the QIAquick PCR Purification Kit (Qiagen) and cloned into a pMD20 vector according to the manufacturer’s instructions (Takara, Dalian, China). Tenfold dilution series of the recombinant plasmid of pMD20-CMNV was prepared for the construction of a standard curve and evaluation of the detection limit of the TaqMan RT-qPCR assay. Analytical specificity of the TaqMan RT-qPCR assay The specificity of the CMNV TaqMan RT-qPCR assay was tested using the nucleic acid of Taura syndrome virus (TSV), yellow head virus (YHV), monodon baculovirus (MBV), white spot syndrome virus (WSSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), and hepatopancreatic parvovirus (HPV) as templates, which were preserved in the authors’ laboratory. The CMNV RNA and total RNAs of the healthy shrimp were used as positive and negative controls. The TaqMan RT-qPCR reaction was run in triplicate for each template. Standard curve and analytical sensitivity of the TaqMan RT-qPCR assay The plasmid pMD20-CMNV containing the target fragment from the CMNV RdRp gene was used as a template to generate a standard curve to determine the sensitivity of the TaqMan RT-qPCR assay. Tenfold serial dilutions (5.7 × 108 to 5.7 copies) and a non-template control were tested in duplicate. The standard curve was generated from the measured Ct values (y) against the logarithmic concentration of the plasmid of pMD20-CMNV (x) using BIORAD CFX96 Software (Version 6.0.14) and Microsoft Excel. Meanwhile, the total RNA was extracted from CMNV-infected P. vannamei and a 10-fold serial dilution of the total RNA (9.6 pg × 103 to 100) was used as the template for TaqMan RT-qPCR to determine the analytical sensitivity of this TaqMan RT-qPCR assay under the optimal conditions. The detection sensitivity of the TaqMan RT-qPCR was determined based on the highest dilution that resulted in a detectable amplification signal. Evaluation of the TaqMan RT-qPCR assay on clinical samples The this TaqMan RT-qPCR assay was used to determine the presence of CMNV in 225 clinical samples. Total RNA of the samples was extracted and used as templates, and the optimized CMNV TaqMan RT-qPCR was performed as described above. Simultaneously, a reference TaqMan RT-PCR assay (named as Pooljun (2016) assay in the following text) previously described by Pooljun (2016) was performed on the same total RNA samples. The calculation of diagnostic sensitivity (DSe) and diagnostic specificity (DSp; World Organization for Animal Health, 2017) between 4

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the two methods were based on the following formula: DSe = True positive cases (TP) / (TP + False negative cases (FN)) and DSp = True negative cases (TN) / (TN + False positive cases (FP)). Repeatability of the TaqMan RT-qPCR assay The inter- and intra-assay repeatability of the TaqMan RT-qPCR assay was assessed in triplicate via tenfold dilution series of the 5.7×108–5.7×101 copies/μL pMD20-CMNV of template. The coefficient of variation (CV) in the intra-assay was evaluated in three independent runs with the eight tenfold dilution series of pMD20-CMNV. Inter-assay variability was also confirmed using the same templates in three runs conducted over three days. Repeatability was evaluated using the CV, which is defined as the percentage of the standard deviation (SD) to the mean of Ct for each of the different pMD20-CMNV dilutions in this study. The analysis of variance (ANOVA) was performed using one-way, completely randomized design in SPSS® (version 13, SPSS Inc., Chicago, IL, USA).

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Results Optimization of TaqMan PCR assay Optimization of temperature and duration of the reverse transcription for the TaqMan RT-qPCR assay showed that the lowest threshold cycle (Ct) was observed with an incubation duration of 15 min at 50.8 ºC for the reverse transcription of RNA template (Fig.2 a and b). Likewise, amplification occurred earlier and with the lowest standard deviation (SD) of 0.14 at an annealing temperature of 52.7 ºC (Fig.2 c). The lowest average Ct value (20.26) was produced when the concentration of primers was set as 0.4 μM, but with a high and non-acceptable SD value (0.37), indicating substantial fluctuation of amplification efficiency. The lowest SD value (0.07) of the Ct was produced when the concentration of primers was set as 0.2 μM with the second lowest average Ct value (20.34). The lowest average Ct value (17.92) was produced when the concentration of probe was set to 0.5 μM with a high and non-acceptable SD value (0.32). The lowest SD value (0.09) of the Ct was produced when the concentration of probe was set as 0.2 μM with the second lowest average Ct value (18.43). Therefore, both of the optimal concentration of primers and probe were set as 0.2 μM finally in consideration of the stability of the TaqMan RT-qPCR and the cost economy of the reagents (Fig.2 d and e). Optimization of the concentration of Enzyme Mix II showed that the highest fluorescence and the lowest threshold cycle (Fig.2 f) were observed with 0.5 μL of the Enzyme Mix II. Therefore, all subsequent TaqMan PCR mixtures for CMNV detection comprised 0.5 μL of the Enzyme Mix II, 12.5 μL 2× One Step RT-PCR Buffer III, 0.2 μM each of forward primer and reverse primer, 0.2 μM probe, 1 μL template, and nuclease-free water to a final reaction volume of 25 μL. Analytical specificity of TaqMan RT-qPCR assay for CMNV Positive results were only obtained when CMNV RNA was used as template. RNA or DNA from other viruses, including TSV, YHV, MBV, WSSV, IHHNV, and HPV, was not amplified by the TaqMan RT-qPCR assay (Fig. 3). The results indicate that the TaqMan RT-qPCR is specific for amplification of CMNV RNA. 5

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Standard curve of the TaqMan RT-qPCR assay A linear relationship between the starting template concentration of plasmid of pMD20-CMNV and the Ct value was observed over nine orders magnitude in a 10× dilution series (Fig. 4 a and b). A standard curve was generated from the threshold cycle values (y) against the logarithmic concentration of plasmid and resulted in the formula of the standard curve, Ct=-3.130 log (starting quantity, Sq) + 40.893 (r2 = 0.996). Based on this equation, the amplification efficiency of the TaqMan RT-qPCR was calculated to be 108.7%. Analytical sensitivity of the TaqMan RT-qPCR assay When the plasmid was used as template, the lowest concentration with a detectable amplification signal was 5.7 copies pMD20-CMNV (Fig. 4 a). When total RNA was used as template, the TaqMan RT-qPCR assay was capable of detecting as little as 9.6 pg of total RNA from CMNV-infected shrimp within 40 cycles (Fig. 5). Repeatability of TaqMan RT-qPCR assay Using template concentrations ranging from 5.7×108–5.7×101 copies/μL, the CMNV TaqMan RT-qPCR assay showed good repeatability, with intra- and inter-assay CV less than 2.27% and 2.32%, respectively (Table 1). The results of analysis of variance showed that the P values of inter-assay variation for each concentration gradient of templates were >0.05, indicating the mean inter-assay Ct differences were not significant (Table 2). Clinical application of TaqMan RT-qPCR assay Total RNA from 225 clinical samples were used to compare the newly developed TaqMan RT-qPCR assay with the previously reported Pooljun (2016) assay. The previously reported Pooljun (2016) assay results indicated that 26 of the 225 samples were positive for CMNV. This TaqMan RT-qPCR assay showed that 25 samples from the 26 CMNV-positive samples determined by previously reported Pooljun (2016) assay gave positive results. Moreover, four out of the 199 samples that were CMNV negative according to the Pooljun (2016) assay yielded a positive result with this TaqMan RT-qPCR assay (Table 3). Therefore, the DSe and DSp values for this TaqMan RT-qPCR method compared with the previously reported Pooljun (2016) assay were 96.2% and 98.0% (Table 4), respectively.

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Discussion For the detection of shrimp viruses such as CMNV, histopathological analysis, coupled with reverse transcription PCR confirmation are the traditional standard despite the relatively long period of time necessary to obtain results (Zhang et al., 2014; Erickson et al., 2002; Tang et al., 2007; Tang et al., 2011). In this study, we developed a quick and specific method for CMNV detection and quantitation. This method combines reverse transcription and amplification in one step, amplifies target nucleic acids efficiently, and is a valuable and novel diagnostic tool for the detection of CMNV. The variation of RdRp genes of different CMNV isolates were tracked in a previous study, revealing that sequences from different isolates had a 0% to 4% difference at the nucleotide sequence level compared to the original CMNV RdRp 6

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gene (GenBank access: KM112247; Zhang et al., 2017). Thus to ensure the newly developed detection method was suitable for different isolates of the target virus, the primers were designed to selectively amplify a conserved region within the genomes of all known genotypes of interest (Rose et al., 2003; Zlateva et al., 2011; Choudhary et al., 2014 Cassar et al., 2017). In so doing, the designed set of TaqMan RT-qPCR primers and probe target a 198 bp region (nucleotide position from 886 to 1083) of the original CMNV RdRp genes and can theoretically work well for the detection of all known CMNV isolates. To validate the newly developed assay, 225 clinical samples were tested simultaneously using both the newly developed TaqMan RT-qPCR and the assay developed by Pooljun (2016). This comparison revealed a discrepancy in five samples. Four out of the five samples were CMNV negative using the Pooljun (2016) assay but positive with this assay. When the amplicons of these four samples were sequenced, twenty sites within the CMNV target RdRp gene had single base mutations, and two mutation sites corresponded to nucleotides 1093 and 1110 of the original CMNV RdRp gene, which are the first and the last nucleic acid bases of reverse primer of Pooljun (2016) assay, respectively (data not shown). Thus it is likely that the two mutations in the CMNV variant from the four samples lead to the detection failure using the Pooljun (2016) assay. Evaluation of the tissues from these four shrimp with in situ hybridization (ISH) and transmission electron microscope (TEM) indicated that the four samples were indeed infected by CMNV (data not shown). The 5th sample of JC170316011, be determined as CMNV positive by Pooljun (2016) assay and CMNV negative by both this TaqMan RT-qPCR and the RT-nPCR, was also proved to be mild infection with CMNV by analysis of ISH and TEM (Data not shown). The authors tried to clone the CMNV target fragment of RdRp gene of the sample of JC170316011 by CMNV RT-nPCR, but the RT-nPCR assay of JC170316011 generated a negative result. Low viral copies in the sample of JC170316011 might be the reason of result in the failure detection of this TaqMan RT-qPCR. Results from this study show that the primers and probe designed in this study can detect all currently known CMNV variants. Nevertheless, the genomes of RNA viruses have characteristically high mutation rates, as does the RNA polymerase that lacks proofreading activity (Steinhauer et al., 1992; Ashfaq et al., 2011; Smith et al., 2013; Sexton et al., 2016), so attention should be continuously given to the genetic variation of CMNV in the future. If new variation of CMNV genomic RNA is detected in the future, evaluation of the validity of the primers and probe should be conducted to ensure the reliability of current CMNV detection methods. In the report of Pooljun (2016) assay, cDNA synthesis was done by first incubating at 42 °C for 30 min for reverse transcription and then incubating at 85 °C for 5 min to inactivate the reverse transcriptase. In the newly developed assay, this time consuming step has been removed by combining the reverse transcription and amplification into one step. The conventional reverse transcription nested PCR (RT-nPCR) methods, including the CMNV RT-nPCR assay (Zhang et al., 2014), usually needs about 4-6 h, and can detect as few as 10 to 100 copies (Billam et al., 2008; Tong et al., 2008; La et al., 2014; Kembou et al., 2017) of the target CMNV 7

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gene. The total detection time was 50 min of CMNV RT-LAMP assay and its detection limit was 27 copies of the target plasmid in the previous report (Zhang et al., 2017). The one-step TaqMan RT-qPCR assay established in this study can be completed in only 60 min and can detect as few as 5.7 copies of the target gene. That is, comparing to previously reported Pooljun (2016) assay, and the conventional method of CMNV RT-nPCR assay, and the CMNV RT-LAMP assay, the TaqMan RT-qPCR method is more sensitive. Comparing to previously reported Pooljun (2016) assay, and the conventional method of CMNV RT-nPCR assay, the TaqMan RT-qPCR method is more time effective. The quantitative reverse transcription loop-mediated isothermal amplification (qRT-LAMP) assay for CMNV was reported recently (Zhang et al., 2017). An equation of quantitation calculated with the high correlation coefficient (r2 = 0.9953) was established in that study and worked well for quantification of CMNV when the starting template was in the range of 108 to 103 copies. However, when the copy number of starting template decreased to less than 1000 copies, the correlation coefficient decreased significantly, which is in accordance with the results from previous reports of LAMP assay. Several reports show that it is difficult to determine the exact correlation between the initial template quantity when the starting template is in very low concentrations using LAMP assays (Mori et al., 2004; Suzuki et al., 2011; Wei et al., 2013). In contrast, when the newly developed TaqMan RT-qPCR assay was used to analyze the starting templates ranging from 5.7×108–5.7×101 copies, it showed a high correlation coefficient (r2 =0.996) good repeatability (i.e., intraand inter-assay CV were less than 2.27% and 2.32%, respectively). Thus, this TaqMan RT-qPCR assay is more appropriate than qRT-LAMP as a quantitation tool, especially when the starting template is lower than 1000 copies. In conclusion, the present study describes a rapid one step, highly sensitive, and highly specific TaqMan RT-qPCR method for the detection of CMNV. This assay provides an alternative approach for the routine detection and quantification of CMNV that will be especially useful in the absence of histopathological experience.

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Conflict of interest The authors have declared no conflict of interest.

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Ethical approval This article does not contain any studies with human participants performed by any of the authors, and ethical approval was given by the institute ethics committee. Acknowledgements The authors would like to thank Dr. Thomas P. Loch in Michigan State University for his generous help in the grammatical correction of the manuscript. This work was supported by the Central Public-interest Scientific Institution Basal Research Fund， CAFS (NO. 2017HY-ZD0301), the Central Public-interest Scientific Institution Basal Research Fund, YSFRI, CAFS (NO.20603022015003), the National Natural Science Foundation of China (31672695), the Projects of International Exchange and 8

Cooperation in Agriculture, the Ministry of Agriculture and Rural Affairs of China-Science, the Technology and Innovation Cooperation in Aquaculture with Tropical Countries along the Belt and Road, and the Project on Introduction of International Advanced Agriculture Science and Technology of the Ministry of Agriculture (Grant: 2016-X56), the Construction Programme for “Taishan Scholarship” of Shandong Province of China. References Ashfaq, U.A., Javed, T., Rehman, S., Nawaz, Z., Riazuddin, S., 2011. An overview of HCV molecular

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detection of human astrovirus. J. Virol. Methods 188: 126–131. World Organization for Animal Health (OIE), Principles and methods of validation of diagnostic assays for infectious diseases. In: Manual of Diagnostic Tests for Aquatic Animals, OIE. Paris, France Chapter 1.1.2 (2017).

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Xing, H., 2004. Discussion of the control measures for the “bottom death” (covert mortality disease) of Pacific white shrimp. China Fish 4: 88–89. (in Chinese with English abstract).

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Xu, Z., Ji, F., 2009. Comprehensive control of the covert mortality disease of Pacific white shrimp. (Chinese J) Fish Guide to be Rich 1: 60. (in Chinese with English abstract)

Zhang, Q.H., 2004. To be cautious of “bottom death” in the intensive farming of Pacific white shrimp. (Chinese J) Sci. Fish Farming 10: 48–49. (in Chinese with English abstract)

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Zhang, Q.L., Liu, Q., Liu, S., Yang, H.L., Liu, S., Zhu, L.L., Yang, B., Jin, J.T., Ding, L.L., Wang, X.H., Liang, Y., Wang, Q.T., Huang, J., 2014. A new nodavirus is associated with covert mortality disease of shrimp. J. Gen. Virol. 95: 2700–2709.

Zhang, Q.L., Liu, S., Yang, H.L., Zhu, L.L., Wan, X.H., Li, X.P., Huang, J., 2017. Reverse transcription loop-mediated isothermal amplification for rapid and quantitative assay of covert mortality nodavirus in shrimp. J. Invertebr. Pathol. 150: 130–135. Zhang, Q.L., Xu, T.T., Wan, X.Y., Liu, S., Wang, X.H., Li, X.P., Dong, X., Yang, B., Huang, J., 2017. Prevalence and distribution of covert mortality nodavirus (CMNV) in cultured crustacean. Virus Res. 233: 113–119. 10

Zlateva, K.T., Crusio, K.M., Leontovich, A.M., Lauber, C., Claas, E., Kravchenko, A.A., Spaan, W.J., Gorbalenya, A.E., 2011. Design and validation of consensus-degenerate hybrid oligonucleotide primers for broad and sensitive detection of corona- and toroviruses. J. Virol. Methods 177: 174–

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183.

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Figure captions

Figure 1

P

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F

SC R

P-F

P-P

P-R

U

R

Fig. 1. Target gene of the TaqMan RT-qPCR for covert mortality nodavirus (CMNV) and

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alignments of the target gene from different CMNV isolate. CMNV indicated the target gene of

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CMNV from the orignal isolate and CMNV_Variant indicated the target gene of CMNV from the

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variant isolate. GenBank accession numbers of CMNV and CMNV_Variant were KM112247and MH728993, respectively. Nucleotide sequences used for primer design are indicated by arrows. F:

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forward primer; R: reverse primer; P: probe; P-F: forward primer of Pooljun (2016) assay (Pooljun

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et al., 2016); P-R: reverse primer of Pooljun (2016) assay; P-P: probe of Pooljun (2016) assay.

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Figure 2

Fig. 2. Optimization of the TaqMan RT-qPCR reaction for the detection of the RNA-dependent

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RNA polymerase gene of covert mortality nodavirus (CMNV). (a) Effect of temperature for reverse transcription on the TaqMan RT-qPCR reaction. (b) Effect of time for reverse transcription on the TaqMan RT-qPCR reaction. (c) Effect of annealing temperature on the TaqMan RT-qPCR

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reaction. (d) Effect of concentration of primers on the TaqMan RT-qPCR reaction. (e) Effect of concentration of probe on the TaqMan RT-qPCR reaction. (f) Effect of volumes of PrimeScript RT Enzyme Mix II on the TaqMan RT-qPCR reaction.

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Figure 3

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Fig. 3. Analytical specificity of the covert mortality nodavirus (CMNV) TaqMan RT-qPCR assay.

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The number of 1indicates the three amplification plots of CMNV; The number of 2 indicates the three amplification plots of Taura syndrome virus; The number of 3 indicates the three

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amplification plots of yellow head virus; The number of 4 indicates the three amplification plots of

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monodon baculovirus; The number of 5 indicates the three amplification plots of white spot syndrome virus; The number of 6 indicates the three amplification plots of infectious hypodermal and hematopoietic necrosis virus; The number of 7 indicates the three amplification plots of

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hepatopancreatic parvovirus. The number of 8 indicates the three amplification plots of the

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negative control. The expected amplification plot of CMNV is indicated by the arrow.

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Figure 4

Fig. 4. The amplification plots and standard curve of the 10-fold serial dilutions of plasmid DNA

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by the covert mortality nodavirus (CMNV) TaqMan RT-qPCR. (a) The amplification plots of

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TaqMan RT-qPCR with serial 10-fold dilutions of the plasmid. Copies of pMD20-CMNV plasmid:

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5.7×108, 5.7×107, 5.7×106, 5.7×105, 5.7×104, 5.7×103, 5.7×102, 5.7×101, and 5.7×100 copies (from left to right). (b) Standard curve and standard curve equation for the CMNV-specific TaqMan

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pMD20-CMNV DNA and Ct value.

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RT-qPCR assay generated from the amplification plots between the serial 10-fold diluted

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Figure 4

Fig. 5. Analytical sensitivity or limits of detection of covert mortality nodavirus (CMNV)-positive

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total RNA from Litopenaeus vannamei by the diagnostic TaqMan RT-qPCR for CMNV.

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Amplification plots 1–5 (from left to right): reaction conducted using 10-fold serial dilutions of

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RNA from L. vannamei: 9.6 × 103 pg, 9.6 × 102 pg, 9.6 × 101 pg, and 9.6× 100 pg, respectively;

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Amplification plot 5 was the negative control (NC).

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Table

Table 1. Intra-assay and inter-assay variability of TaqMan RT-qPCR assay Dilution of plasmid (copies/reaction )

Intra-assay Ct

Inter-assay Ct

SD

CV (%)

Mean

SD

CV (%)

16.30

0.10

0.60

16.29

0.07

0.44

7

20.28

0.07

0.33

19.89

0.40

2.02

6

5.7×10

23.55

0.17

0.73

23.49

0.27

1.15

5.7×105

27.31

0.16

0.57

26.97

0.42

1.56

4

5.7×10

29.85

0.10

0.32

29.76

0.21

5.7×103

33.52

0.16

0.48

33.37

0.26

5.7×102

38.08

0.86

2.27

37.09

0.86

5.7×101

39.62

0.54

1.36

39.79

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5.7×10

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5.7×10

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Mean

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0.74

0.71 0.78 2.32 1.87

Table 2. Analysis of variance of inter-assay variability of TaqMan RT-qPCR assay F value

P value

5.7×108

0.23

1.91

5.7×107

0.71

0.38

5.7×106

0.55

0.69

5.7×105

0.42

1.07

4

0.78

0.27

3

0.97

0.04

2

0.77

0.28

1

/

/

5.7×10 5.7×10 5.7×10

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5.7×10

Inter-assay Ct

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Dilution of plasmid (copies/reaction )

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Table 3. Comparison of results of the newly developed TaqMan RT-qPCR assay and Pooljun’s (2016) TaqMan RT-PCR assay performed with 225 clinical samples of living shrimp samples (The sample numbers of merely CMNV positive samples were listed). The TaqMan RT-qPCR copies/μL

Pooljun’s (2016) TaqMan RT-PCR

JC170104001

4.49×102

＋

JC170104002

7.32×10

2

＋

2.59×10

2

＋

2.40×10

3

＋

1.29×10

3

＋

2.92×10

3

＋

3.20×10

1

＋

2.73×10

2

＋

JC170225011

2.28×10

2

JC170225012

8.62

JC170111004 JC170225009 JC170225010

JC170225014

9.19×104

JC170225015

2.04×105

JC170225016

2.38×103

JC170312002

2.42

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＋ ＋ ＋

3.58×10

－

－

＋

6.7

＋

JC170317003

JC170317005

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JC170317006 JC170320001

a

JC170320002 JC170320003

a

JC170322015 JC170324005 JC170324007

＋

9.88

a

JC170317004

1

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JC170317002

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＋

b

JC170317001

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7

＋

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JC170316011

＋

a

JC170316010

a

＋

2.90×10

1.99×10

＋

2

JC170225013

JC170313001

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JC170111003

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JC170111002

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JC170105002

1

－

2.21×10

1

＋

9.95×10

1

＋

3.30×10

1

＋

1

－

2

＋

3.54×10

1.81×10 1.31×10

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Sample number

－

9.96

＋

6.98 1.02×10

2

＋

3.37×10

2

＋

Notice sample numbers JC170316010, JC170317003, JC170320001, and JC170320003 were

negative by Pooljun (2016) assay but tested positive by the newly developed TaqMan RT-qPCR. b

Notice sample numbers JC170316011was positive by Pooljun (2016) assay but tested

negative by the newly developed TaqMan RT-qPCR. 19

Table 4. The comparison of detection results of the newly developed TaqMan RT-qPCR and Pooljun’s (2016) TaqMan RT-PCR Number of reference samples Positive samples, 26

Negative samples,199

Detected positive

25

4

Detected negative

1

195

Dse=25/26×100% =96.2%

Dsp=195/199×100% =98.0%

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Calculation

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