|
|
|
| Establishment and Application of HRM Assay for Detection of Enzootic nasal tumor virus 2 (ENTV-2) in Goat (Capra hircus) |
| ZHANG Jing-Peng1, JIANG Jin-Xiu1, LIN Yu-Sheng1, YOU Wei1, LIU Dao-Quan2, MAO Kun-Min3, JIANG Bin1, HU Qi-Lin1,* |
1 Institute of Animal Husbandry & Veterinary Medicine, Fujian Academy of Agricultural Sciences, Fuzhou 350013, China;
2 Fuzhou Center for Animal Disease Prevention and Control, Fuzhou 350026, China;
3 Fuqing Animal Husbandry and Veterinary Center, Fuqing 350300, China |
|
|
|
|
Abstract For the detection of enzootic nasal tumor (ENT) caused by Enzootic nasal tumor virus 2 (ENTV-2), the RT-PCR followed by restriction enzyme digestion method, RT-PCR, and qPCR had been established. However, the existence of endogenous retroviruses (ERVs) can interfere with the accuracy of the results. In this study, ENTV-2 was compared with ERVs and Jaagsiekte sheep retrovirus (JSRV) by bioinformatics methods, the conserved sequence of envelope protein (env) gene of ENTV-2 was found, and a pair of specific primers were designed. By optimizing the reaction conditions, a high resolution melting curve (HRM) detection method was established. The optimization results showed that the best reaction system was 0.4 μmol/L upstream and downstream primers, 1 μL template, 1 μL styo9 (2.5 μmol/L), 2×Premix Taq 10 μL, add ddH2O to a final volume of 20 μL. The reaction condition was 94 ℃ for 2 min; 94 ℃ for 15 s; 59 ℃ for 15 s, 72 ℃ for 15 s, 40 cycles. The env gene recombinant plasmid was constructed as standard positive plasmid and used to determine the specificity, sensitivity, and reproducibility of the HRM method. The results showed that the established HRM method could only specifically detected ENTV-2 but not ERVs which was highly homologous with ENTV-2. The sensitivity of the HRM was 82.7 copies/μL for the positive plasmid. The repeatablity of the method was good, the coefficient variations (CV) of intra-batch and inter-batch were < 1%.The positive detection rate of 92 clinical samples was 18.7%, and the coincidence rate with TaqMan detection method was 100%. This study provides technical support for the early and rapid detection of the diseases related to ENT.
|
|
Received: 11 January 2022
|
|
|
|
Corresponding Authors:
* hql562713@163.com
|
|
|
|
[1] 崔明全, 吴辰斌, 汪洋, 等. 2015. 耐氟喹诺酮类药物空肠弯曲菌HRM快速检测方法的建立[J]. 中国兽医科学, 45(5): 441-447.
(Cui M Q, Wu C B, Wang Y, et al.2015. Establishment of a high-resolution melting assay for detection of fluoroquinolones-resistant Campylobacter jejuni[J]. Chinese Veterinary Science, 45(5): 441-447.)
[2] 贾广乐, 周莉, 林祥梅, 等. 2013. 奶牛白细胞黏附缺陷症HRM检测方法的建立及应用[J]. 中国兽医科学, (12): 1274-1279.
(Jia G L , Zhou L, Lin X M, et al. 2013. Development and application of high resolution melting analysis for detection of BLAD alleles[J]. Chinese Veterinary Science, (12): 1274-1279.)
[3] 江锦秀, 林裕胜, 江斌, 等. 2017. 福建山羊地方性鼻内肿瘤的分子流行病学调查[J]. 福建农业学报, 32(8): 837-841.
(Jiang J X, Lin Y S, Jiang B, et al.2017. Molecular epidemiology of Enzootic nasal tumor virus on goats in Fujian[J]. Fujian Journal of Agricultural Sciences, 32(8): 837-841.)
[4] 刘志成, 贾伟新, 孙敏华, 等. 2016. H7N9亚型禽流感病毒HRM检测方法的建立[J]. 中国兽医科学, 46(11): 1388-1393.
(Liu Z C, Jia W X, Sun M H, et al.2016. Development of HRM method for detection of H7N9 subtype Avian influenza virus[J]. Chinese Veterinary Science, 46(11): 1388-1393.)
[5] 权自芳, 叶泥, 文彩芳, 等. 2015. 地方性鼻内腺瘤中DNMTs启动子甲基化状态及转录表达的研究[J]. 中国预防兽医学报, 37(5): 353-356.
(Quan Z F, Ye Ni, Wen C F, et al.2015. Promoter methylation and the mRNA expression of DNA methyltransferases gene in Enzootic nasal tumor[J]. Chinese Journal of Preventive Veterinary Medicine, 37(5): 353-356.)
[6] 王景, 周曼, 何亚鹏, 等. 2017. 一起山羊地方性鼻内肿瘤的诊断[J]. 动物医学进展, 38(2): 129-131.
(Wang J, Zhou M, He Y P, et al.2017. Diagnosis of enzootic nasal tumor in goats[J]. Progress in Veterinary Medicine, 38(2): 129-131.)
[7] Apostolidi E D, Psalla D, Chassalevris T, et al.2019. Development of real-time PCR-based methods for the detection of Enzootic nasal tumor virus 2 in goats[J]. Archives of Virology, 164(3): 707-716.
[8] Cousens C, Minguijon E, GARCIA M, et al.1996. PCR-based detection and partial characterization of a retrovirus associated with contagious intranasal tumors of sheep and goats[J]. Journal of Virology, 70(11): 7580-7583.
[9] Heras M, Jalon J, Sharp J M.1991. Pathology of enzootic intranasal tumor in thirty-eight goats[J]. Veterinary Pathology, 28(6): 474-481.
[10] De las Heras M, Garcia de Jalon J A, Minguijon E, et al.1995. Experimental transmission of enzootic intranasal tumors of goats[J]. Veterinary Pathology, 32(1): 19-23.
[11] Geng Y, Wang K Y, Yang Q G, et al.2010. Descriptive study of enzootic nasal adenocarcinoma in goats in southwestern China[J]. Transboundary and Emerging Diseases, 57(3): 197-200.
[12] He Y P, Zhang Q, Wang J, et al.2017. Full-length genome sequence analysis of Enzootic nasal tumor virus isolated from goats in China[J]. Virology Journal, 14(1): 141-152.
[13] Herrmann M G, Durtschi J D, Wittwer C T, et al.2007. Expanded instrument comparison of amplicon DNA melting analysis for mutation scanning and genotyping[J]. Clinical Chemistry, 53(8): 1544-1548.
[14] Loukopoulos P, Giadinis N D, Psychas V, et al.2010. Enzootic nasal tumour of goats in Greece[J]. Journal of Comparative Pathology, 143(4): 343-350.
[15] Palmarini M, Mura M, Spencer T E.2004. Endogenous betaretroviruses of sheep: Teaching new lessons in retroviral interference and adaptation[J]. Journal of General Virology, 85(Pt 1): 1-13.
[16] Monis P T, Giglio S, Saint C P, et al.2005. Comparison of SYTO9 and SYBR GreenⅠfor real-time polymerase chain reaction and investigation of the effect of dye concentration on amplication and DNA melting curve analysis[J]. Analytical Biochemistry, 340(1): 24-34.
[17] Reed G H, Wittwer C T.2004. Sensitivity and specificity of single-nucleotide polymorphism scanning by high-resolution melting analysis[J]. Clinical Chemistry, 50(10): 1748-1754.
[18] Scocco P, Mariotti F, Ceccarelli P, et al.2001. Origin of enzootic intranasal tumor in the goat (Capra hircus): A glycohistochemical approach[J]. Veterinary Pathology, 38(1): 98-104.
[19] Tesfaye R C, Tirumala B K, Settypalli T B K, et al.2019. An HRM assay to differentiate sheeppox virus vaccine strains from sheeppox virus field isolates and other Capripoxvirus species[J]. Nature, 9(1): 1-8.
[20] Vitellozzi G, Mughetti L, Palmarini M, et al.2010. Enzootic intranasal tumour of goats in Italy[J]. Journal of Veterinary Medicine, 40(7): 459-468.
[21] Wittwer C T, Reed G H, Gundry C N, et al.2003. High-resolution genotyping by amplicon melting analysis using LCGreen[J]. Clinical Chemistry, 49(6): 853-860. |
|
|
|
