- Email: [email protected]
A TaqMan probe based real-time PCR for the detection of Decapod iridescent virus 1
Journal of Invertebrate Pathology 173 (2020) 107367
Contents lists available at ScienceDirect
Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip
Short Communication
A TaqMan probe based real-time PCR for the detection of Decapod iridescent virus 1
T
Liang Qiua, Xing Chena, Xiao-Meng Guoa,b, Wen Gaoa,c, Ruo-Heng Zhaoa, Qing-Li Zhanga, ⁎ Bing Yanga, Jie Huanga,b,c, a Laboratory for Marine Fisheries Science and Food Production Processes, Pilot National Laboratory for Marine Science and Technology (Qingdao), Key Laboratory of Maricultural Organism Disease Control, Ministry of Agriculture and Rural Affairs, Qingdao Key Laboratory of Mariculture Epidemiology and Biosecurity, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China b School of Marine Science and Engineering, Qingdao Agricultural University, Qingdao 266109, China c Shanghai Ocean University, Shanghai 201306, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Decapod iridescent virus 1 Shrimp hemocyte iridescent virus Cherax quadricarinatus iridovirus Real-time PCR Shrimp
Decapod iridescent virus 1 (DIV1) was proven to be the aetiological agent of a disease causing mass die-offs of shrimp, prawn and crayfish. The specific purpose of this study was to develop a new sensitive real-time PCR method for the specific detection of DIV1. A pair of primers that amplify a 142 bp fragment and a TaqMan probe were selected for the major capsid protein gene of DIV1. They were shown to be specific for DIV1 and did not react with other common shrimp pathogens or healthy shrimp DNA. The method could detect as virus levels as low as 1.2 copies of DIV1 plasmid DNA.
1. Introduction An emerging disease has caused high mortality and severe losses of farmed shrimp, prawn and crayfish in China. The aetiological agent of the new disease is a double-stranded DNA virus named Decapod iridescent virus 1 (DIV1) (Chen et al., 2019a; Qiu et al., 2017; Qiu et al., 2019a; Xu et al., 2016). DIV1 was first described by Xu et al. (2016) as Cherax quadricarinatus iridovirus (CQIV) and by Qiu et al. (2017) as Shrimp hemocyte iridescent virus (SHIV). Clinical signs include hepatopancreatic atrophy with colour fading, empty stomach and guts, and a reddish coloured body (Qiu et al., 2017). Diseased Macrobrachium rosenbergii exhibit a typical white area under the carapace at the base of rostrum called ‘white head’ disease or ‘white spot’ disease (Qiu, et al., 2019a). Moribund individuals sink to the bottom of deep water where dead individuals accumulate, and the cumulative mortality may reach 80%. DIV1 has been reported in some coastal provinces of China since 2014, including Zhejiang, Guangdong and Hebei Provinces (Qiu et al., 2017). Target surveillance in China in 2017–2018 revealed that DIV1 was detected in 11 of 16 provinces (Qiu et al., 2018c; Qiu et al., 2019b). DIV1 is an enveloped icosahedral virus with a diameter of about 150 nm and a double-stranded DNA genome of about 166 K bp (Li et al., 2017; Qiu et al., 2018b). Based on two original isolations, SHIV 20141215 and CQIV CN01 (ICTV, 2019), the virus was assigned by the International Committee on Taxonomy of Viruses (ICTV) as the only ⁎
member of the new genus Decapodiridovirus within the family Iridoviridae. Currently known susceptible species include C. quadricarinatus, Penaeus vannamei, Macrobrachium nipponense, M. rosenbergii, Procambarus clarkii and Exopalaemon carinicauda (Chen et al., 2019a; Qiu et al., 2017; Qiu et al., 2019a; Xu et al., 2016). Two species of crab, Eriocheir sinensis and Pachygrapsus crassipes also were infected with DIV1 in experimental challenge by intramuscular injection (Pan et al., 2017). It is worth noting that positive results have been detected by PCR in samples of Penaeus chinensis, Penaeus japonicus, Macrobrachium superbum, Nereis succinea or some cladocera (Qiu et al., 2017; Qiu et al., 2018c; Qiu et al., 2019a; Qiu et al., 2019b). Among the detection methods, TaqMan probe based real-time PCR is the most sensitive and specific method for detecting and quantifying shrimp viruses. Some real-time PCR methods can detect one copy of viral DNA or cDNA for several shrimp viruses such as Hepatopancreatic parvovirus (HPV), Penaeus monodon baculovirus (MBV) and Infectious myonecrosis virus (IMNV) (Yan et al., 2009, 2010; Liu et al., 2013). A TaqMan probe based real-time PCR assay was established to detect ATPase gene of DIV1 in our previous research (Qiu et al., 2018a). This method was specific and the detection limit was four copies per reaction. However, the amplified fragment using this method overlapped with the only published nested PCR method for DIV1 (Qiu et al., 2017), which might have been due to cross-contamination when both methods were used in the laboratory. To avoid this risk, we describe a new real-
Corresponding author at: Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, #106, Nanjing Road, Qingdao 266071, China. E-mail address: [email protected] (J. Huang).
https://doi.org/10.1016/j.jip.2020.107367 Received 23 December 2019; Received in revised form 17 March 2020; Accepted 22 March 2020 Available online 03 April 2020 0022-2011/ © 2020 Elsevier Inc. All rights reserved.
Journal of Invertebrate Pathology 173 (2020) 107367
L. Qiu, et al.
Fig. 1. A: Analytic specificity test. Templates were DNA extracted from shrimp infected with 1. DIV1, 2. WSSV, 3. IHHNV, 4. VAHPND, 5. EHP, 6. Healthy shrimp; and 7. Water (NTC). B: Analytic sensitivity test. 1–9: 1.2 × 108 to 1.2 copies μl−1 DIV1 DNA; 10: water (NTC). C: The standard curve.
temperature of 60 °C for 30 s. Amplification, detection, and data analysis were performed using a CFX-96 Quantitative Fluorescence Instrument (Bio-Rad, USA).
time PCR assay for the detection of major capsid protein (MCP) of DIV1. 2. Materials and methods
2.4. Construction of positive control vectors and standards for quantification
2.1. Sample collection and DNA extraction P. vannamei were collected from farms in Guangdong and Shandong Province of China and stored at −80 °C. Total DNA was extracted from 30 mg of shrimp cephalothorax tissue using a TIANamp Marine Animal DNA Kit (TIANGEN Biotech, Beijing, China) according to the manufacturer’s instructions.
The 142-bp DNA fragment was cloned into the pMD18-T vector (TaKaRa, Dalian, China). The sequence of the insert was confirmed by sequencing (Sangon Biotech, Shanghai, China). The concentration of the plasmid was then determined using a NanoDrop 2000 (Thermo Fisher, America). The copy number of the plasmid containing the 142bp insert was estimated, and a series of dilutions were prepared as standards.
2.2. Primers and probe Specific primers and a probe were designed for the DIV1 MCP gene (GenBank accession No. KY681039, MF599468, NC_040612) (Qiu et al., 2017; Qiu et al., 2018b; Li et al., 2017) using AlleleID version 5.01 (PREMIER Biosoft). The upstream (142F 5′- AAT CCA TGC AAG GTT CCT CAG G -3′) and downstream (142R 5′- CAA TCA ACA TGT CGC GGT GAA C -3′) primers yielded a PCR amplicon of 142 bp. The TaqMan probe (5′- CCA TAC GTG CTC GCT CGG CTT CGG -3′) was synthesized and labeled with 6-carboxyfluorescein (FAM) on the 5′ end and with N,N,N′,N′-tetramethyl-6-carboxyrhododamine (TAMRA) on the 3′ end.
2.5. Analytic specificity and analytic sensitivity To test the analytic specificity, DNA extracted from shrimps infected with white spot syndrome virus (WSSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), Vibrio causing acute hepatopancreatic necrosis disease (VAHPND) and Enterocytozoon hepatopenaei (EHP), were used as the template in the real-time PCR assay. The negative, positive and blank controls were the DNA of healthy P. vannamei, DNA of P. vannamei infected with DIV1, and water, respectively. Each assay was conducted in triplcate. The analytic sensitivity of the real-time PCR was determined using a series of 10-fold dilutions of purified plasmid pDIV1. The concentration of plasmid DNA ranged from 1.2 × 100 to 1.2 × 108 copies per reaction. Each assay was conducted in triplcate.
2.3. Real-time PCR assay and procedure Real-time PCR reactions were performed in a 20-μL reaction system consisting of 10 μL 2 × Master Mix, 500 nM of each primer (142F/ 142R), 200 nM TaqMan probe and 1 μL DNA template using FastStart Essential DNA Probes Master (Roche). Briefly, after an initial denaturation step at 95 °C for 10 min, amplifications were carried out with 40 cycles at a melting temperature of 95 °C for 10 s and an annealing
2.6. Clinical sample test A total of 300 DNA samples were extracted from the cephalothorax 2
Journal of Invertebrate Pathology 173 (2020) 107367
L. Qiu, et al.
Acknowledgements
Table 1 Detection results of samples with unknown infection status by two real-time PCR. Comparison of two methods
Positive
Positive2 Negative2 Total
141 4 145
1
Negative 2 153 155
1
This work was supported by the Marine S&T Fund of Shandong Province for Pilot National Laboratory for Marine Science and Technology (Qingdao) (2018SDKJ0502-3); the National Key R&D Program of China (2019YFD0900101); China Postdoctoral Science Foundation (2019M650170); the Postdoctoral innovation project of Shandong Province (201902043); the Postdoctoral Applied Research Foundation of Qingdao (2019); the China Agriculture Research System (CARS-47).
Total 143 157 300
Note: Subscript 1 = result tested using the real-time PCR developed by Qiu et al., 2018a; Subscript 2 = result tested using the real-time PCR developed in this study.
References of P. vannamei with unknown DIV1 infection status. Extracted DNA was tested by the real-time PCR developed in this study and previously (Qiu et al., 2018a). Diagnostic sensitivity (DSe) and diagnostic specificity (DSp) were determined according to the World Organisation for Animal Heath (OIE) manual (OIE, 2019).
Chen, X., Qiu, L., Wang, H.L., Zou, P.Z., Dong, X., Li, F.H., Huang, J., 2019a. Susceptibility of Exopalaemon carinicauda to the infection with Shrimp hemocyte iridescent virus (SHIV 20141215), a strain of Decapod iridescent virus 1 (DIV1). Viruses 11 (4), 387. https://doi.org/10.3390/v11040387. Chen, Z.W., Huang, J., Zhang, F., Zhou, Y., Huang, H.J., 2019b. Detection of shrimp hemocyte iridescent virus by recombinase polymerase amplification assay. Mol. Cell. Probes (in press). doi: 10.1016/j.mcp.2019.101475. Durand, S.V., Lightner, D.V., 2002. Quantitative real time PCR for the measurement of white spot syndrome virus in shrimp. J. Fish Dis. 25, 381–389. ICTV, 2019. One New Genus with One New Species in the Subfamily Betairidovirinae. Available online: https://talk.ictvonline.org/files/ictv_official_taxonomy_updates_ since_the_8th_report/m/animal-dna-viruses-and-retroviruses/8051. Li, F., Xu, L., Yang, F., 2017. Genomic characterization of a novel iridovirus from redclaw crayfish Cherax quadricarinatus: evidence for a new genus within the family Iridoviridae. J. Gen. Virol. 98 (10), 2589–2595. https://doi.org/10.1099/jgv.0. 000904. Liu, H.L., Yan, D.C., Sun, H.S., Wang, Y.Y., Wang, L., 2013. A real-time PCR for the detection of infectious myonecrosis virus in penaeid shrimp. J. Invertebr. Pathol. 113, 237–239. https://doi.org/10.1016/j.jip.2013.04.009. OIE, 2019. Manual of diagnostic tests for aquatic animals. World Organisation for Animal Health, http://www.oie.int/en/international-standard-setting/aquatic-manual/ access-online/. Pan, C.K., Yuan, H.F., Wang, T.T., Yang, F., Chen, J.M., 2017. Study of Cherax quadricarinatus iridovirus in two crab. J. Appl. Oceanogr. 36 (1), 82–86 (in Chinese). Qiu, L., Chen, M.M., Wan, X.Y., Li, C., Zhang, Q.L., Wang, R.Y., Cheng, D.Y., Dong, X., Yang, B., Wang, X.H., Xiang, J.H., Huang, J., 2017. Characterization of a new member of Iridoviridae, Shrimp hemocyte iridescent virus (SHIV), found in white leg shrimp (Litopenaeus vannamei). Sci. Rep. 7 (1), 11834. https://doi.org/10.1038/ s41598-017-10738-8. Qiu, L., Chen, M.M., Wan, X.Y., Zhang, Q.L., Li, C., Dong, X., Yang, B., Huang, J., 2018a. Detection and quantification of Shrimp hemocyte iridescent virus by TaqMan probe based real-time PCR. J. Invertebr. Pathol. 154, 95–101. https://doi.org/10.1016/j. jip.2018.04.005. Qiu, L., Chen, M.M., Wang, R.Y., Wan, X.Y., Li, C., Zhang, Q.L., Dong, X., Yang, B., Xiang, J.H., Huang, J., 2018b. Complete genome sequence of shrimp hemocyte iridescent virus (SHIV) isolated from white leg shrimp, Litopenaeus vannamei. Arch. Virol. 163 (3), 781–785. https://doi.org/10.1007/s00705-017-3642-4. Qiu, L., Dong, X., Wan, X.Y., Huang, J., 2018c. Analysis of iridescent viral disease of shrimp (SHID) in 2017. In Analysis of Important Diseases of Aquatic Animals in China in 2017 (in Chinese). Fishery and Fishery Administration Bureau under the Ministry of Agriculture and Rural Affairs, National Fishery Technical Extension Center, Eds., China Agriculture Press, Beijing, pp. 187-204, ISBN 978-7-109-24522-8. Qiu, L., Chen, X., Zhao, R.H., Li, C., Gao, W., Zhang, Q.L., Huang, J., 2019a. Description of a natural infection with Decapod iridescent virus 1 in farmed giant freshwater prawn, Macrobrachium rosenbergii. Viruses 11 (4), 354. https://doi.org/10.3390/v11040354. Qiu, L., Dong, X., Wan, X.Y., Huang, J., 2019b. Analysis of iridescent viral disease of shrimp (SHID) in 2018. In Analysis of Important Diseases of Aquatic Animals in China in 2018 (in Chinese). Fishery and Fishery Administration Bureau under the Ministry of Agriculture and Rural Affairs, National Fishery Technical Extension Center, Eds., China Agriculture Press, Beijing, pp. 189-207, ISBN 978-7-109-25963-8. Xu, L., Wang, T., Li, F., Yang, F., 2016. Isolation and preliminary characterization of a new pathogenic iridovirus from redclaw crayfish Cherax quadricarinatus. Dis. Aquat. Organ. 120 (1), 17–26. https://doi.org/10.3354/dao03007. Yan, D.C., Tang, K.F.J., Lightner, D.V., 2009. Development of a real-time PCR assay for detection of monodon baculovirus (MBV) in penaeid shrimp. J. Invertebr. Pathol. 102, 97–100. Yan, D.C., Tang, K.F.J., Lightner, D.V., 2010. A real-time PCR for the detection of hepatopancreatic parvovirus (HPV) of penaeid shrimp. J. Fish Dis. 33, 507–511.
3. Results and discussion The amplification plot showed that only DNA extracted from cephalothorax of DIV1-infected shrimp (Fig. 1A, curve 1) was detected; DNA was not detected from samples of healthy shrimp or shrimps infected with WSSV, IHHNV, VAHPND and EHP (Fig. 1A, curves 2–6). Therefore, the analytical specificity test showed that the real-time PCR assay developed in this study was specific to DIV1. The sensitivity test revealed that the real-time PCR assay could detect DIV1 DNA as low as 1.2 copies/reaction (Fig. 1B), more sensitive than the real-time PCR methods for DIV1 developed previously (Qiu et al., 2018a) or for WSSV (Durand and Lightner, 2002). The standard curve showed a high correlation coefficient (R2 = 0.997) within the range of 1.2 × 101–1.2 × 108 DNA copies/reaction. The regressive equation was Ct = −3.169·lg(DIV1 DNA copies) + 42.524 (Fig. 1C). Therefore, this new real-time PCR assay could be used as an appropriate quantitative tool within the range of 1.2 × 101–1.2 × 108 DNA copies/ reaction. Results of two real-time PCR methods for DIV1 were in agreement that 141 samples were positive and 153 samples were negative, and 6 samples showed different results for these two real-time PCR methods (Table 1). Based on the above test results, the DSe (the proportion of samples from known infected reference animals that were positive in an assay) and the DSp (the proportion of samples from known uninfected reference animals that were negative in an assay) of the new real-time PCR were 97.2% and 98.7%, respectively. In this study, we developed a specific and highly sensitive TaqMan based real-time PCR method for DIV1. Since the viral load in larvae or juveniles could be very low, the sensitivity of the detection method is crucial. Among the published detection methods for DIV1, the new realtime PCR is by far the most sensitive (Qiu et al., 2017; Qiu et al., 2018a; Chen et al., 2019b). Also, it avoids the risk of cross-contamination when used with the published nested PCR method due to targeting different genes. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
3
Contents lists available at ScienceDirect
Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip
Short Communication
A TaqMan probe based real-time PCR for the detection of Decapod iridescent virus 1
T
Liang Qiua, Xing Chena, Xiao-Meng Guoa,b, Wen Gaoa,c, Ruo-Heng Zhaoa, Qing-Li Zhanga, ⁎ Bing Yanga, Jie Huanga,b,c, a Laboratory for Marine Fisheries Science and Food Production Processes, Pilot National Laboratory for Marine Science and Technology (Qingdao), Key Laboratory of Maricultural Organism Disease Control, Ministry of Agriculture and Rural Affairs, Qingdao Key Laboratory of Mariculture Epidemiology and Biosecurity, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China b School of Marine Science and Engineering, Qingdao Agricultural University, Qingdao 266109, China c Shanghai Ocean University, Shanghai 201306, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Decapod iridescent virus 1 Shrimp hemocyte iridescent virus Cherax quadricarinatus iridovirus Real-time PCR Shrimp
Decapod iridescent virus 1 (DIV1) was proven to be the aetiological agent of a disease causing mass die-offs of shrimp, prawn and crayfish. The specific purpose of this study was to develop a new sensitive real-time PCR method for the specific detection of DIV1. A pair of primers that amplify a 142 bp fragment and a TaqMan probe were selected for the major capsid protein gene of DIV1. They were shown to be specific for DIV1 and did not react with other common shrimp pathogens or healthy shrimp DNA. The method could detect as virus levels as low as 1.2 copies of DIV1 plasmid DNA.
1. Introduction An emerging disease has caused high mortality and severe losses of farmed shrimp, prawn and crayfish in China. The aetiological agent of the new disease is a double-stranded DNA virus named Decapod iridescent virus 1 (DIV1) (Chen et al., 2019a; Qiu et al., 2017; Qiu et al., 2019a; Xu et al., 2016). DIV1 was first described by Xu et al. (2016) as Cherax quadricarinatus iridovirus (CQIV) and by Qiu et al. (2017) as Shrimp hemocyte iridescent virus (SHIV). Clinical signs include hepatopancreatic atrophy with colour fading, empty stomach and guts, and a reddish coloured body (Qiu et al., 2017). Diseased Macrobrachium rosenbergii exhibit a typical white area under the carapace at the base of rostrum called ‘white head’ disease or ‘white spot’ disease (Qiu, et al., 2019a). Moribund individuals sink to the bottom of deep water where dead individuals accumulate, and the cumulative mortality may reach 80%. DIV1 has been reported in some coastal provinces of China since 2014, including Zhejiang, Guangdong and Hebei Provinces (Qiu et al., 2017). Target surveillance in China in 2017–2018 revealed that DIV1 was detected in 11 of 16 provinces (Qiu et al., 2018c; Qiu et al., 2019b). DIV1 is an enveloped icosahedral virus with a diameter of about 150 nm and a double-stranded DNA genome of about 166 K bp (Li et al., 2017; Qiu et al., 2018b). Based on two original isolations, SHIV 20141215 and CQIV CN01 (ICTV, 2019), the virus was assigned by the International Committee on Taxonomy of Viruses (ICTV) as the only ⁎
member of the new genus Decapodiridovirus within the family Iridoviridae. Currently known susceptible species include C. quadricarinatus, Penaeus vannamei, Macrobrachium nipponense, M. rosenbergii, Procambarus clarkii and Exopalaemon carinicauda (Chen et al., 2019a; Qiu et al., 2017; Qiu et al., 2019a; Xu et al., 2016). Two species of crab, Eriocheir sinensis and Pachygrapsus crassipes also were infected with DIV1 in experimental challenge by intramuscular injection (Pan et al., 2017). It is worth noting that positive results have been detected by PCR in samples of Penaeus chinensis, Penaeus japonicus, Macrobrachium superbum, Nereis succinea or some cladocera (Qiu et al., 2017; Qiu et al., 2018c; Qiu et al., 2019a; Qiu et al., 2019b). Among the detection methods, TaqMan probe based real-time PCR is the most sensitive and specific method for detecting and quantifying shrimp viruses. Some real-time PCR methods can detect one copy of viral DNA or cDNA for several shrimp viruses such as Hepatopancreatic parvovirus (HPV), Penaeus monodon baculovirus (MBV) and Infectious myonecrosis virus (IMNV) (Yan et al., 2009, 2010; Liu et al., 2013). A TaqMan probe based real-time PCR assay was established to detect ATPase gene of DIV1 in our previous research (Qiu et al., 2018a). This method was specific and the detection limit was four copies per reaction. However, the amplified fragment using this method overlapped with the only published nested PCR method for DIV1 (Qiu et al., 2017), which might have been due to cross-contamination when both methods were used in the laboratory. To avoid this risk, we describe a new real-
Corresponding author at: Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, #106, Nanjing Road, Qingdao 266071, China. E-mail address: [email protected] (J. Huang).
https://doi.org/10.1016/j.jip.2020.107367 Received 23 December 2019; Received in revised form 17 March 2020; Accepted 22 March 2020 Available online 03 April 2020 0022-2011/ © 2020 Elsevier Inc. All rights reserved.
Journal of Invertebrate Pathology 173 (2020) 107367
L. Qiu, et al.
Fig. 1. A: Analytic specificity test. Templates were DNA extracted from shrimp infected with 1. DIV1, 2. WSSV, 3. IHHNV, 4. VAHPND, 5. EHP, 6. Healthy shrimp; and 7. Water (NTC). B: Analytic sensitivity test. 1–9: 1.2 × 108 to 1.2 copies μl−1 DIV1 DNA; 10: water (NTC). C: The standard curve.
temperature of 60 °C for 30 s. Amplification, detection, and data analysis were performed using a CFX-96 Quantitative Fluorescence Instrument (Bio-Rad, USA).
time PCR assay for the detection of major capsid protein (MCP) of DIV1. 2. Materials and methods
2.4. Construction of positive control vectors and standards for quantification
2.1. Sample collection and DNA extraction P. vannamei were collected from farms in Guangdong and Shandong Province of China and stored at −80 °C. Total DNA was extracted from 30 mg of shrimp cephalothorax tissue using a TIANamp Marine Animal DNA Kit (TIANGEN Biotech, Beijing, China) according to the manufacturer’s instructions.
The 142-bp DNA fragment was cloned into the pMD18-T vector (TaKaRa, Dalian, China). The sequence of the insert was confirmed by sequencing (Sangon Biotech, Shanghai, China). The concentration of the plasmid was then determined using a NanoDrop 2000 (Thermo Fisher, America). The copy number of the plasmid containing the 142bp insert was estimated, and a series of dilutions were prepared as standards.
2.2. Primers and probe Specific primers and a probe were designed for the DIV1 MCP gene (GenBank accession No. KY681039, MF599468, NC_040612) (Qiu et al., 2017; Qiu et al., 2018b; Li et al., 2017) using AlleleID version 5.01 (PREMIER Biosoft). The upstream (142F 5′- AAT CCA TGC AAG GTT CCT CAG G -3′) and downstream (142R 5′- CAA TCA ACA TGT CGC GGT GAA C -3′) primers yielded a PCR amplicon of 142 bp. The TaqMan probe (5′- CCA TAC GTG CTC GCT CGG CTT CGG -3′) was synthesized and labeled with 6-carboxyfluorescein (FAM) on the 5′ end and with N,N,N′,N′-tetramethyl-6-carboxyrhododamine (TAMRA) on the 3′ end.
2.5. Analytic specificity and analytic sensitivity To test the analytic specificity, DNA extracted from shrimps infected with white spot syndrome virus (WSSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), Vibrio causing acute hepatopancreatic necrosis disease (VAHPND) and Enterocytozoon hepatopenaei (EHP), were used as the template in the real-time PCR assay. The negative, positive and blank controls were the DNA of healthy P. vannamei, DNA of P. vannamei infected with DIV1, and water, respectively. Each assay was conducted in triplcate. The analytic sensitivity of the real-time PCR was determined using a series of 10-fold dilutions of purified plasmid pDIV1. The concentration of plasmid DNA ranged from 1.2 × 100 to 1.2 × 108 copies per reaction. Each assay was conducted in triplcate.
2.3. Real-time PCR assay and procedure Real-time PCR reactions were performed in a 20-μL reaction system consisting of 10 μL 2 × Master Mix, 500 nM of each primer (142F/ 142R), 200 nM TaqMan probe and 1 μL DNA template using FastStart Essential DNA Probes Master (Roche). Briefly, after an initial denaturation step at 95 °C for 10 min, amplifications were carried out with 40 cycles at a melting temperature of 95 °C for 10 s and an annealing
2.6. Clinical sample test A total of 300 DNA samples were extracted from the cephalothorax 2
Journal of Invertebrate Pathology 173 (2020) 107367
L. Qiu, et al.
Acknowledgements
Table 1 Detection results of samples with unknown infection status by two real-time PCR. Comparison of two methods
Positive
Positive2 Negative2 Total
141 4 145
1
Negative 2 153 155
1
This work was supported by the Marine S&T Fund of Shandong Province for Pilot National Laboratory for Marine Science and Technology (Qingdao) (2018SDKJ0502-3); the National Key R&D Program of China (2019YFD0900101); China Postdoctoral Science Foundation (2019M650170); the Postdoctoral innovation project of Shandong Province (201902043); the Postdoctoral Applied Research Foundation of Qingdao (2019); the China Agriculture Research System (CARS-47).
Total 143 157 300
Note: Subscript 1 = result tested using the real-time PCR developed by Qiu et al., 2018a; Subscript 2 = result tested using the real-time PCR developed in this study.
References of P. vannamei with unknown DIV1 infection status. Extracted DNA was tested by the real-time PCR developed in this study and previously (Qiu et al., 2018a). Diagnostic sensitivity (DSe) and diagnostic specificity (DSp) were determined according to the World Organisation for Animal Heath (OIE) manual (OIE, 2019).
Chen, X., Qiu, L., Wang, H.L., Zou, P.Z., Dong, X., Li, F.H., Huang, J., 2019a. Susceptibility of Exopalaemon carinicauda to the infection with Shrimp hemocyte iridescent virus (SHIV 20141215), a strain of Decapod iridescent virus 1 (DIV1). Viruses 11 (4), 387. https://doi.org/10.3390/v11040387. Chen, Z.W., Huang, J., Zhang, F., Zhou, Y., Huang, H.J., 2019b. Detection of shrimp hemocyte iridescent virus by recombinase polymerase amplification assay. Mol. Cell. Probes (in press). doi: 10.1016/j.mcp.2019.101475. Durand, S.V., Lightner, D.V., 2002. Quantitative real time PCR for the measurement of white spot syndrome virus in shrimp. J. Fish Dis. 25, 381–389. ICTV, 2019. One New Genus with One New Species in the Subfamily Betairidovirinae. Available online: https://talk.ictvonline.org/files/ictv_official_taxonomy_updates_ since_the_8th_report/m/animal-dna-viruses-and-retroviruses/8051. Li, F., Xu, L., Yang, F., 2017. Genomic characterization of a novel iridovirus from redclaw crayfish Cherax quadricarinatus: evidence for a new genus within the family Iridoviridae. J. Gen. Virol. 98 (10), 2589–2595. https://doi.org/10.1099/jgv.0. 000904. Liu, H.L., Yan, D.C., Sun, H.S., Wang, Y.Y., Wang, L., 2013. A real-time PCR for the detection of infectious myonecrosis virus in penaeid shrimp. J. Invertebr. Pathol. 113, 237–239. https://doi.org/10.1016/j.jip.2013.04.009. OIE, 2019. Manual of diagnostic tests for aquatic animals. World Organisation for Animal Health, http://www.oie.int/en/international-standard-setting/aquatic-manual/ access-online/. Pan, C.K., Yuan, H.F., Wang, T.T., Yang, F., Chen, J.M., 2017. Study of Cherax quadricarinatus iridovirus in two crab. J. Appl. Oceanogr. 36 (1), 82–86 (in Chinese). Qiu, L., Chen, M.M., Wan, X.Y., Li, C., Zhang, Q.L., Wang, R.Y., Cheng, D.Y., Dong, X., Yang, B., Wang, X.H., Xiang, J.H., Huang, J., 2017. Characterization of a new member of Iridoviridae, Shrimp hemocyte iridescent virus (SHIV), found in white leg shrimp (Litopenaeus vannamei). Sci. Rep. 7 (1), 11834. https://doi.org/10.1038/ s41598-017-10738-8. Qiu, L., Chen, M.M., Wan, X.Y., Zhang, Q.L., Li, C., Dong, X., Yang, B., Huang, J., 2018a. Detection and quantification of Shrimp hemocyte iridescent virus by TaqMan probe based real-time PCR. J. Invertebr. Pathol. 154, 95–101. https://doi.org/10.1016/j. jip.2018.04.005. Qiu, L., Chen, M.M., Wang, R.Y., Wan, X.Y., Li, C., Zhang, Q.L., Dong, X., Yang, B., Xiang, J.H., Huang, J., 2018b. Complete genome sequence of shrimp hemocyte iridescent virus (SHIV) isolated from white leg shrimp, Litopenaeus vannamei. Arch. Virol. 163 (3), 781–785. https://doi.org/10.1007/s00705-017-3642-4. Qiu, L., Dong, X., Wan, X.Y., Huang, J., 2018c. Analysis of iridescent viral disease of shrimp (SHID) in 2017. In Analysis of Important Diseases of Aquatic Animals in China in 2017 (in Chinese). Fishery and Fishery Administration Bureau under the Ministry of Agriculture and Rural Affairs, National Fishery Technical Extension Center, Eds., China Agriculture Press, Beijing, pp. 187-204, ISBN 978-7-109-24522-8. Qiu, L., Chen, X., Zhao, R.H., Li, C., Gao, W., Zhang, Q.L., Huang, J., 2019a. Description of a natural infection with Decapod iridescent virus 1 in farmed giant freshwater prawn, Macrobrachium rosenbergii. Viruses 11 (4), 354. https://doi.org/10.3390/v11040354. Qiu, L., Dong, X., Wan, X.Y., Huang, J., 2019b. Analysis of iridescent viral disease of shrimp (SHID) in 2018. In Analysis of Important Diseases of Aquatic Animals in China in 2018 (in Chinese). Fishery and Fishery Administration Bureau under the Ministry of Agriculture and Rural Affairs, National Fishery Technical Extension Center, Eds., China Agriculture Press, Beijing, pp. 189-207, ISBN 978-7-109-25963-8. Xu, L., Wang, T., Li, F., Yang, F., 2016. Isolation and preliminary characterization of a new pathogenic iridovirus from redclaw crayfish Cherax quadricarinatus. Dis. Aquat. Organ. 120 (1), 17–26. https://doi.org/10.3354/dao03007. Yan, D.C., Tang, K.F.J., Lightner, D.V., 2009. Development of a real-time PCR assay for detection of monodon baculovirus (MBV) in penaeid shrimp. J. Invertebr. Pathol. 102, 97–100. Yan, D.C., Tang, K.F.J., Lightner, D.V., 2010. A real-time PCR for the detection of hepatopancreatic parvovirus (HPV) of penaeid shrimp. J. Fish Dis. 33, 507–511.
3. Results and discussion The amplification plot showed that only DNA extracted from cephalothorax of DIV1-infected shrimp (Fig. 1A, curve 1) was detected; DNA was not detected from samples of healthy shrimp or shrimps infected with WSSV, IHHNV, VAHPND and EHP (Fig. 1A, curves 2–6). Therefore, the analytical specificity test showed that the real-time PCR assay developed in this study was specific to DIV1. The sensitivity test revealed that the real-time PCR assay could detect DIV1 DNA as low as 1.2 copies/reaction (Fig. 1B), more sensitive than the real-time PCR methods for DIV1 developed previously (Qiu et al., 2018a) or for WSSV (Durand and Lightner, 2002). The standard curve showed a high correlation coefficient (R2 = 0.997) within the range of 1.2 × 101–1.2 × 108 DNA copies/reaction. The regressive equation was Ct = −3.169·lg(DIV1 DNA copies) + 42.524 (Fig. 1C). Therefore, this new real-time PCR assay could be used as an appropriate quantitative tool within the range of 1.2 × 101–1.2 × 108 DNA copies/ reaction. Results of two real-time PCR methods for DIV1 were in agreement that 141 samples were positive and 153 samples were negative, and 6 samples showed different results for these two real-time PCR methods (Table 1). Based on the above test results, the DSe (the proportion of samples from known infected reference animals that were positive in an assay) and the DSp (the proportion of samples from known uninfected reference animals that were negative in an assay) of the new real-time PCR were 97.2% and 98.7%, respectively. In this study, we developed a specific and highly sensitive TaqMan based real-time PCR method for DIV1. Since the viral load in larvae or juveniles could be very low, the sensitivity of the detection method is crucial. Among the published detection methods for DIV1, the new realtime PCR is by far the most sensitive (Qiu et al., 2017; Qiu et al., 2018a; Chen et al., 2019b). Also, it avoids the risk of cross-contamination when used with the published nested PCR method due to targeting different genes. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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