Letters in Applied Microbiology ISSN 0266-8254
ORIGINAL ARTICLE
Development of a rapid cyprinid herpesvirus 2 detection method by loop-mediated isothermal amplification L.-G. Liang1, J. Xie1 and D. Luo1,2 1 Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi, China 2 College of Animal Science & Technology, Gansu Agricultural University, Lanzhou, China
Significance and Impact of the Study: Herpesviral haematopoietic necrosis, caused by cyprinid herpesvirus 2 (CyHV-2), is a severe disease of goldfish and Prussian carp associated with high mortality. We developed a loop-mediated isothermal amplification (LAMP) assay to detect CyHV-2 at relatively low plasmid DNA copy levels. The results show that the LAMP assay has a number of advantages (simple, sensitive, rapid and specific) over the conventional polymerase chain reaction and can be applied in the laboratory and field. Particularly, the method is highly applicable to facilitate surveillance and early diagnosis of CyHV-2.
Keywords Carassius auratus gibelio, cyprinid herpesvirus 2, loop-mediated isothermal amplification, PCR, rapid detection. Correspondence Jun Xie, Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081, China. E-mail: [email protected] 2013/2519: received 16 December 2013, revised 10 June 2014 and accepted 11 June 2014 doi:10.1111/lam.12296
Abstract Cyprinid herpesvirus 2 (CyHV2) is a pathogen that causes severe disease and high mortality in goldfish and Prussian carp. We developed a six primer loopmediated isothermal amplification (LAMP) assay targeting the intercapsomeric triplex protein gene. CyHV-2 DNA was 10-fold serially diluted (108– 100 copies ll1) and was used as the template to determine primer sensitivity. LAMP assays were performed with DNA templates from other pathogens to determine specificity. The LAMP assay had an unequivocal detection limit of 10 copies ll1, which was 100 times lower than that of the polymerase chain reaction. Other pathogen strains were not amplified by the LAMP primers, indicating good specificity. SYBR Green I was added to visually detect the amplification products. Assay applicability was evaluated in 120 samples of Carassius auratus gibelio, and a positive rate of 925% was obtained. In conclusion, a conventional LAMP assay has high convenience, rapidity, sensitivity and specificity for detecting CyHV-2 in infected aquatic organisms.
Introduction The DNA virus cyprinid herpesvirus 2 (CyHV-2), also known as herpesviral haematopoietic necrosis virus or goldfish haematopoietic necrosis virus, is an important pathogen of Prussian carp, Carassius auratus gibelio (Tomas et al. 2012; Wang et al. 2012) and goldfish, Carassius auratus (Groff et al. 1998; Jung et al., 1995). First detected in C. auratus gibelio in Hungary (Doszpoly et al. 2011), this virus has also been reported in ornamental goldfish in Japan, where it caused severe haematopoietic necrosis and high mortality in the spring seasons of 1992 and 1993 (Waltzek et al. 2005). Similar disease outbreaks 432
have been reported in goldfish in Taiwan (Chang et al. 1999), Australia (Stephens et al. 2004), the United Kingdom (Jeffrey et al. 2007) and the United States of America (Goodwin et al. 2009). CyHV-2 has caused significant economic losses in cultured Prussian carp in China (Wang et al. 2012; Luo et al. 2013; Xu et al. 2013). To date, no sensitive cell line is available for isolation (Gilad et al. 2004; Jeffrey et al. 2007). Conventional polymerase chain reaction (PCR) and real-time PCR with high sensitivity and specificity have been developed (Goodwin et al. 2006a; b; Waltzek et al. 2009). The loop-mediated isothermal amplification (LAMP) assay is a novel gene amplification method with high specificity, sensitivity and
Letters in Applied Microbiology 59, 432--437 © 2014 The Society for Applied Microbiology
L.-G. Liang et al.
Establishment of LAMP for the detection of CyHV-2
rapidity and can be applied for disease diagnosis in aquaculture. The method is performed under isothermal conditions with a set of four specially designed primers that recognize six distinct sequences of the target DNA (Notomi et al. 2000). The purpose of this study was to develop a LAMP assay to rapidly detect CyHV-2 in host organisms. Result and discussion Optimizing the LAMP assay conditions LAMP products obtained using 1 ll of nucleic acids from diseased fish were turbid, fluoresced green under UV light, formed a white pellet in the reaction tube and revealed the typical ladder-like pattern on agarose gel electrophoresis, which is a characteristic of LAMP. None of the four manifestations (turbidity, fluorescence, pellets and the ladder-like pattern) were observed in the virusfree negative control (Fig. 1). A 60-min LAMP reaction with CyHV-2 specific primers at 60–66°C produced many bands of different sizes by agarose gel electrophoresis. Amplification of CyHV-2 resulted in a ladder-like pattern, whereas the PCR product was a specific DNA band. Because CyHV-2 LAMP produced brighter and more distinct bands at 63°C than any other temperature, 63°C was selected as the optimal temperature (data not shown). No product was amplified at reaction times
ORIGINAL ARTICLE
Development of a rapid cyprinid herpesvirus 2 detection method by loop-mediated isothermal amplification L.-G. Liang1, J. Xie1 and D. Luo1,2 1 Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi, China 2 College of Animal Science & Technology, Gansu Agricultural University, Lanzhou, China
Significance and Impact of the Study: Herpesviral haematopoietic necrosis, caused by cyprinid herpesvirus 2 (CyHV-2), is a severe disease of goldfish and Prussian carp associated with high mortality. We developed a loop-mediated isothermal amplification (LAMP) assay to detect CyHV-2 at relatively low plasmid DNA copy levels. The results show that the LAMP assay has a number of advantages (simple, sensitive, rapid and specific) over the conventional polymerase chain reaction and can be applied in the laboratory and field. Particularly, the method is highly applicable to facilitate surveillance and early diagnosis of CyHV-2.
Keywords Carassius auratus gibelio, cyprinid herpesvirus 2, loop-mediated isothermal amplification, PCR, rapid detection. Correspondence Jun Xie, Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081, China. E-mail: [email protected] 2013/2519: received 16 December 2013, revised 10 June 2014 and accepted 11 June 2014 doi:10.1111/lam.12296
Abstract Cyprinid herpesvirus 2 (CyHV2) is a pathogen that causes severe disease and high mortality in goldfish and Prussian carp. We developed a six primer loopmediated isothermal amplification (LAMP) assay targeting the intercapsomeric triplex protein gene. CyHV-2 DNA was 10-fold serially diluted (108– 100 copies ll1) and was used as the template to determine primer sensitivity. LAMP assays were performed with DNA templates from other pathogens to determine specificity. The LAMP assay had an unequivocal detection limit of 10 copies ll1, which was 100 times lower than that of the polymerase chain reaction. Other pathogen strains were not amplified by the LAMP primers, indicating good specificity. SYBR Green I was added to visually detect the amplification products. Assay applicability was evaluated in 120 samples of Carassius auratus gibelio, and a positive rate of 925% was obtained. In conclusion, a conventional LAMP assay has high convenience, rapidity, sensitivity and specificity for detecting CyHV-2 in infected aquatic organisms.
Introduction The DNA virus cyprinid herpesvirus 2 (CyHV-2), also known as herpesviral haematopoietic necrosis virus or goldfish haematopoietic necrosis virus, is an important pathogen of Prussian carp, Carassius auratus gibelio (Tomas et al. 2012; Wang et al. 2012) and goldfish, Carassius auratus (Groff et al. 1998; Jung et al., 1995). First detected in C. auratus gibelio in Hungary (Doszpoly et al. 2011), this virus has also been reported in ornamental goldfish in Japan, where it caused severe haematopoietic necrosis and high mortality in the spring seasons of 1992 and 1993 (Waltzek et al. 2005). Similar disease outbreaks 432
have been reported in goldfish in Taiwan (Chang et al. 1999), Australia (Stephens et al. 2004), the United Kingdom (Jeffrey et al. 2007) and the United States of America (Goodwin et al. 2009). CyHV-2 has caused significant economic losses in cultured Prussian carp in China (Wang et al. 2012; Luo et al. 2013; Xu et al. 2013). To date, no sensitive cell line is available for isolation (Gilad et al. 2004; Jeffrey et al. 2007). Conventional polymerase chain reaction (PCR) and real-time PCR with high sensitivity and specificity have been developed (Goodwin et al. 2006a; b; Waltzek et al. 2009). The loop-mediated isothermal amplification (LAMP) assay is a novel gene amplification method with high specificity, sensitivity and
Letters in Applied Microbiology 59, 432--437 © 2014 The Society for Applied Microbiology
L.-G. Liang et al.
Establishment of LAMP for the detection of CyHV-2
rapidity and can be applied for disease diagnosis in aquaculture. The method is performed under isothermal conditions with a set of four specially designed primers that recognize six distinct sequences of the target DNA (Notomi et al. 2000). The purpose of this study was to develop a LAMP assay to rapidly detect CyHV-2 in host organisms. Result and discussion Optimizing the LAMP assay conditions LAMP products obtained using 1 ll of nucleic acids from diseased fish were turbid, fluoresced green under UV light, formed a white pellet in the reaction tube and revealed the typical ladder-like pattern on agarose gel electrophoresis, which is a characteristic of LAMP. None of the four manifestations (turbidity, fluorescence, pellets and the ladder-like pattern) were observed in the virusfree negative control (Fig. 1). A 60-min LAMP reaction with CyHV-2 specific primers at 60–66°C produced many bands of different sizes by agarose gel electrophoresis. Amplification of CyHV-2 resulted in a ladder-like pattern, whereas the PCR product was a specific DNA band. Because CyHV-2 LAMP produced brighter and more distinct bands at 63°C than any other temperature, 63°C was selected as the optimal temperature (data not shown). No product was amplified at reaction times
