牦牛轮状病毒VP6基因序列分析及RT-PCR检测方法的建立与应用

doi:10.11843/j.issn.0366-6964.2016.07.021

Abstract

Abstract:

Rotavirus (RV) VP6 gene is the main target gene of molecular detection，but the gene point mutations affects the molecular detection of RV.The purpose of this experiment was to establish a detection method of yak RV by RT-PCR method which applied in clinical samples based on the study of genetic variation yak RV VP6.The fragments of yak RV VP6 ranged in 931-1 338 bp were obtained with the primers designed by Prime 5.0 software from 30 samples of yak diarrhea.Sequence analysis showed that，yak RV VP6 genes represented multiple point mutations in the section of 931-1110 bp compared with bovine rotavirus (BRV)，while the section between 1 100-1 338bp was highly conserved.According to this result，the assay has successfully established a method with good specificity and stability.The primer could amplify the specific fragment of yak RV and BRV，with no amplification of other unrelated pathogens.The detection limit of viral nucleic acid of the assays was 0.45 pg•μL-1.With a remarkable detection rate in yak RV，the RT-PCR method provided a useful tool for the diagnosis and epidemiological investigation of yak RV disease.Additionally，this method can also be used in BRV detection.The RV detection rate in 234 samples of yak diarrhea from different provinces were 60.00% in Tibet (36/60)，95.00% in Qinghai (57/60)，85.19% in Sichuan(46/54) and 90.00% in Yunnan (54/60)，respectively.The result shows that RV infection is an important cause of yak diarrhea.

Key words: yak, rotavirus, VP6 gene, point mutation, RT-PCR

CLC Number:

ZHOU Fang，YUE Hua，ZHANG Bin，LI Fan，CHEN Xi，TANG Cheng. Establishment and Application of an RT-PCR Assay for Yak Rotavirus Based on the Sequence Analysis of Yak Rotavirus VP6 gene[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(7): 1465-1473.

References

［1］BADARACCO A，GARAICOECHEA L，RODRÍGUEZ D，et al．Bovine rotavirus strains circulating in beef and dairy herds in Argentina from 2004 to 2010［J］．Vet Microbiol，2012，158(3-4)：394-399．
［2］MINAMI-FUKUDA F，NAGAI M，TAKAI H，et al.Detection of bovine group A rotavirus using rapid antigen detection kits，RT-PCR and next-generation DNA sequencing［J］.J Vet Med Sci，2013，75(12)：1651-1655.
［3］ALFIERI A F，ALFIERI A A，BARREIROS M A，et al.G and P genotypes of group A rotavirus strains circulating in calves in Brazil 1996-1999［J］.Vet Microbiol，2004，99(3-4)：167-173.
［4］GONZALEZ D D，MOZGOVOJ M V，BELLIDO D，et al.Evaluation of a bovine rotavirus VP6 vaccine efficacy in the calf model of infection and disease［J］.Vet Immunol Immunopathol，2010，137(1-2)：155-160.
［5］WISE A G，SMEDLEY R C，KIUPEL M，et al.Detection of group C rotavirus in juvenile ferrets (Mustela putorius furo) with diarrhea by reverse transcription polymerase chain reaction：sequencing and analysis of the complete coding region of the VP6 gene［J］.Vet Pathol，2009，46(5)：985-991.
［6］STIPP D T，ALFIERI A F，LORENZETTI E，et al.VP6 gene diversity in 11 Brazilian strains of porcine group C rotavirus［J］.Virus Genes，2015，50(1)：142-146.
［7］范晴，谢芝勋，刘加波，等.牛轮状病毒TaqMan实时荧光RT-PCR快速检测方法的建立［J］.中国畜牧兽医，2011，38(4)：105-108.
FAN Q，XIE Z X，LIU J B，et al.Detection of bovine rotavirus by TaqMan based real-time reverse transcription polymerase chain reaction assay［J］.China Animal Husbandry & Veterinary Medicine，2011，38(4)：105-108.(in Chinese)
［8］王洁清.牛轮状病毒RT-PCR检测和基于VP6重组抗原检测血清抗体ELISA方法初步建立［D］.哈尔滨：东北农业大学，2009.
WANG J Q.Detection of bovine rotavirus by RT-PCR and establishment of indirect ELISA to detect bovine rotavirus antibody with VP6 protein［D］.Harbin：Northeast Agricultural University，2009.(in Chinese)
［9］BASERA S S，SINGH R，VAID N，et al.Detection of rotavirus infection in bovine calves by RNA-PAGE and RT-PCR［J］. Indian J Virol，2010，21(2)：144-147.
［10］NYAGA M M，ESONA M D，JERE K C，et al.Genetic diversity of rotavirus genome segment 6(encoding VP6) in Pretoria，South Africa［J］. Springerplus，2014，3：179.
［11］KERIN T K，KANE E M，GLASS R I，et al.Characterization of VP6 genes from rotavirus strains collected in the United States from 1996-2002［J］. Virus Genes，2007，35(3)：489-495.
［12］CHEN X，ZHANG B，YUE H，et al.A novel astrovirus species in the gut of yaks with diarrhea in the Qinghai Tibetan plateau，2013［J］.J Gen Virol，2015.doi：10.1099/gv.0.000303.
［13］ITURRIZA GÓMARA M，WONG C，BLOME S，et al.Molecular characterization of VP6 genes of human rotavirus isolates：correlation of genogroups with subgroups and evidence of independent segregation［J］.J Virol，2002，76(13)：6596-6601.
［14］GAUTAM R，ESONA M D，MIJATOVIC-RUSTEMPASIC S，et al.Real-time RT-PCR assays to differentiate wild-type group A rotavirus strains from Rotarix and Rota Teq vaccine strains in stool samples［J］.Hum Vaccin Immunother，2014，10(3)：767-777.
［15］LIN Y P，KAO C L，CHANG S Y，et al.Determination of human rotavirus VP6 genogroups Ⅰ and Ⅱ by reverse transcription-PCR［J］.J Clin Microbiol，2008，46(10)：3330-3337.
［16］DENNEHY P H.Rotavirus vaccines：an overview［J］.Clin Microbiol Rev，2008，21(1)：198-208.
［17］LAPPALAINEN S，PASTOR A R，MALM M，et al.Protection against live rotavirus chanllenge in mice induced by parenteral and mucosal delivery of VP6 subunit rotavirus vaccine［J］. Arch Virol，2015，160(8)：2075-2078.
［18］MATTHIJNSSENS J，TARAPOREWALA Z F，YANG H，et al.Simian rotaviruses possess divergent gene constellations that originated from interspecies transmission and reassortment［J］.J Virol，2010，84(4)：2013-2026.
［19］MATTHIJNSSENS J，CIARLET M，HEIMAN E，et al.Full genome-based classification of rotaviruses reveals a common origin between human Wa-Like and procine rotavirus strains and human DS-1-like and bovine rotavirus strains［J］.J Virol，2008，82(7)：3204-3219.
［20］DURMAZ R，KALAYCIOGLU A T，ACAR S，et al.Prevalence of rotavirus genotypes in children younger than 5 years of age before the introduction of a universal rotavirus vaccination program：report of rotavirus surveillance in Turkey［J］.PLoS One，2014，9(12)：e113674.
［21］DOAN Y H，NAKAGOMI T，ABOUDY Y，et al.Identification by full-genome analysis of a bovine rotavirus transmitted directly to and causing diarrhea in a human child［J］.J Clin Microbiol，2013，51(1)：182-189.

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Establishment and Application of an RT-PCR Assay for Yak Rotavirus Based on the Sequence Analysis of Yak Rotavirus VP6 gene

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