蜱携带牛丙型肝炎病毒新亚型巢式PCR检测方法的建立

doi:10.11843/j.issn.0366-6964.2022.03.030

畜牧兽医学报 ›› 2022, Vol. 53 ›› Issue (3): 972-977.doi: 10.11843/j.issn.0366-6964.2022.03.030

蜱携带牛丙型肝炎病毒新亚型巢式PCR检测方法的建立

郭銮英, 王妮娜, 李杭远, 纪雨霏, 马骏, 裴明超, 邵建伟, 刘全*

佛山科学技术学院生命科学与工程学院, 佛山 528000

收稿日期:2021-05-18 出版日期:2022-03-23 发布日期:2022-03-31
通讯作者: 刘全,主要从事动物新发疫病研究,E-mail:liuquan@fosu.edu.cn;邵建伟,主要从事动物新发疫病研究,E-mail:jwshao1988@163.com
作者简介:郭銮英(1995-),女,广东潮州人,硕士生,主要从事动物新发疫病研究,E-mail:guoluanyingfs@163.com
基金资助:
广东省珠江人才计划项目(2019CX01 N111);佛山科学技术学院2020年度学生学术基金(xsjj202009kja01)

The Development of Nested PCR Assay for New Subtype of Bovine Hepacivirus in Ticks

GUO Luanying, WANG Nina, LI Hangyuan, JI Yufei, MA Jun, PEI Mingchao, SHAO Jianwei, LIU Quan*

College of Life Sciences and Engineering, Foshan University, Foshan 528000, China

Received:2021-05-18 Online:2022-03-23 Published:2022-03-31

摘要/Abstract

摘要： 旨在提高新发现的牛丙型肝炎病毒毒株——广东湛江毒株(bovine hepacivirus-GDZJ,BovHepV-GDZJ)的检测及诊断能力,初步了解广东地区蜱携带BovHepV-GDZJ的情况,本研究结合宏转录组学、高通量测序及PCR技术,针对高通量测序结果中丰度高的基因区域,设计该病毒的巢式PCR特异性引物。通过优化巢式PCR方法的最佳退火温度,建立检测BovHepV-GDZJ的特异性巢式PCR检测方法。结果显示:本方法对该病毒标准质粒最低检测值为6.8 copies·μL^-1;对沙粒病毒等蜱携带的常见病原均无反应;应用此方法对115份广东湛江的蜱样品进行了检测,测序结果显示,该批蜱携带BovHepV阳性率为46.08%(53/115)。本研究首次在蜱中检测到BovHepV,揭示了BovHepV可能的传播途径,并建立了一套针对BovHepV-GDZJ株的特异性巢式PCR检测方法,为广东地区检测和预防BovHepV提供技术帮助,也为控制BovHepV的流行提供参考。

关键词: 牛丙型肝炎病毒, 巢式PCR, 蜱, 高通量测序

Abstract: A detection method is urgently needed, which not only can improve the detection and diagnostic ability for the newly discovered bovine hepacivirus strain GDZJ, BovHepV-GDZJ, but also contribute to the preliminary understanding of tick carrying BovHepV in Guangdong province. In this study, the specific primers of Nested PCR for BovHepV-GDZJ strain were designed using the gene regions with high abundance in high-throughput sequencing data as templates through macrotranscriptomics, high-throughput sequencing, and PCR. The results showed that the minimum detection value of the method for the standard plasmid containing the gene fragment of BovHepV-GDZJ strain was 6.8 copies·μL^-1, and it had no response to common pathogens carried by ticks such as arenavirus. Moreover, the positive rate was 46.08% (53/115) in the 115 tick samples collected in Zhanjiang, Guangdong. In this study, BovHepV was first detected from ticks, which revealing the possible transmission route of BovHepV. What's more, a nested PCR method for the detection of BovHepV-GDZJ strain was established to provide technical help for the detection and prevention of BovHepV in Guangdong. It also provides some reference value for the control of the HNV epidemic.

Key words: bovine hepacivirus, nested PCR, tick, high-throughput sequencin

中图分类号:

S852.653

郭銮英, 王妮娜, 李杭远, 纪雨霏, 马骏, 裴明超, 邵建伟, 刘全. 蜱携带牛丙型肝炎病毒新亚型巢式PCR检测方法的建立[J]. 畜牧兽医学报, 2022, 53(3): 972-977.

GUO Luanying, WANG Nina, LI Hangyuan, JI Yufei, MA Jun, PEI Mingchao, SHAO Jianwei, LIU Quan. The Development of Nested PCR Assay for New Subtype of Bovine Hepacivirus in Ticks[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(3): 972-977.

参考文献 12

[1]	QIANG X, SHEN X Y, PENG H Y, et al. Complete genome sequence of a novel bovine hepacivirus from Yunnan, China[J]. Arch Virol, 2020, 165(6):1489-1494.
[2]	ELIA G, CARINGELLA F, LANAVE G, et al. Genetic heterogeneity of bovine hepacivirus in Italy[J]. Transbound Emerg Dis, 2020, 67(6):2731-2740.
[3]	TANAKA T, KASAI H, YAMASHITA A, et al. Hallmarks of hepatitis C virus in equine hepacivirus[J]. J Virol, 2014,88(22):13352-13366.
[4]	BURBELO P D, DUBOVI E J, SIMMONDS P, et al. Serology-enabled discovery of genetically diverse hepaciviruses in a new host[J]. J Virol, 2012, 86(11):6171-6178.
[5]	DREXLER J F, CORMAN V M, MULLER M A, et al. Evidence for novel hepaciviruses in rodents[J]. PLoS Pathog, 2013, 9(6):e1003438.
[6]	QUAN P L, FIRTH C, CONTE J M, et al. Bats are a major natural reservoir for hepaciviruses and pegiviruses[J]. Proc Natl Acad Sci U S A, 2013, 110(20):8194-8199.
[7]	LAUCK M, SIBLEY S D, LARA J, et al. A novel hepacivirus with an unusually long and intrinsically disordered NS5A protein in a wild Old World primate[J]. J Virol, 2013, 87(16):8971-8981.
[8]	BAECHLEIN C, FISCHER N, GRUNDHOFF A, et al. Identification of a novel hepacivirus in domestic cattle from Germany[J]. J Virol, 2015, 89(14):7007-7015.
[9]	LU G, OU J J, ZHAO J W, et al. Presence of a novel subtype of bovine hepacivirus in China and expanded classification of bovine hepacivirus strains worldwide into 7 subtypes[J]. Viruses, 2019, 11(9):843.
[10]	BAECHLEIN C, BARON A L, MEYER D, et al. Further characterization of bovine hepacivirus:Antibody response, course of infection, and host tropism[J]. Transbound Emerg Dis, 2019, 66(1):195-206.
[11]	DA SILVA M S, JUNQUEIRA D M, BAUMBACH L F, et al. Comprehensive evolutionary and phylogenetic analysis of hepacivirus N (HNV)[J]. J Gen Virol, 2018, 99(7):890-896.
[12]	HARVEY E, ROSE K, EDEN J S, et al. Extensive diversity of RNA viruses in Australian ticks[J/OL]. J Virol, 2019, 93(3):e01358-18.[2021-10-21]. https://journals.asm.org/doi/epub/10.1128/JVI.01358-18.

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蜱携带牛丙型肝炎病毒新亚型巢式PCR检测方法的建立

The Development of Nested PCR Assay for New Subtype of Bovine Hepacivirus in Ticks

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