基于恒温隔绝式荧光PCR技术现场检测牛轮状病毒方法的建立及应用

doi:10.11843/j.issn.0366-6964.2017.06.014

Abstract

Abstract:

Rotavirus (RV) is an important cause of calves diarrhea. The objective of this study was to establish an insulated isothermal PCR (iiPCR) assay for on-site detecting bovine rotavirus (BRV). A pair of primers and a fluorescent TaqMan probe were designed according to BRV and Yak RV VP6 gene sequences. After optimizing the reaction system and nucleic acid extraction by a commercial PetNAD nucleic acid extraction kit, the iiPCR assay for detecting BRV and yak RV was successfully established. The results showed that the assay could amplify specific fragment of BRV and yak RV, with no amplification of other unrelated pathogens, including bovine coronavirus, bovine astrovirus, bovine viral diarrhea virus, Escherichia coli, Salmonella, Eimeria and Cryptosporidium. The detection limit of viral nucleic acid of the assay was 96.6 copies·μL^-1 with good reproducibility. Thirty clinical diarrhea samples of calves and 30 clinical diarrhea samples of yaks were used to evaluate the coincidence of this assay with two reported assays,and the positive rate of BRV and yak RV samples was 33.33% and 73.33% respectively by this assay,which has 100% coincident detection rate with the reported assays for detecting BRV or Yak RV, respectively. Further, 98.86% positive rate of yak RV was detected by the assay developed in this study in 88 yak diarrhea samples from 14 pastures in Northwest Sichuan plateau in 2016. The established iiPCR assay in this study was specific, sensitive and reproducible, and it can be used for detecting not only BRV but also yak RV. It takes only 1 hour from nucleic acid extraction to report test result with easy operation,which makes it a valuable tool for rapid on-site diagnostics of BRV.

CLC Number:

LI Fan, YUE Hua, QIU Wan-si, ZHANG Bin, HUANG Jian-de, TANG Cheng. Development and Application of an Insulated Isothermal PCR (iiPCR) for On-site Detecting Bovine Rotavirus[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(6): 1092-1098.

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References

[1] PAPP H, LÁSZLÓ B, JAKAB F, et al. Review of group A rotavirus strains reported in swine and cattle[J]. Vet Microbiol, 2013, 165(3-4):190-199.
[2] 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(4):985-991.
[3] 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.
[4] MEBUS C A, UNDERDAHL N R, RHODES M B, et al. Calf diarrhea (scours):reproduced with a virus from a field outbreak[R]. Bulletin:University of Nebraska Agricultural Experiment Station Research, 1969.
[5] CHO Y I, YOON K J. An overview of calf diarrhea-infectious etiology, diagnosis, and intervention[J]. J Vet Sci, 2014, 15(1):1-17.
[6] 李鑫, 常继涛, 刘海军, 等. 我国A群牛轮状病毒感染的血清流行病学调查[J]. 中国预防兽医学报, 2010, 32(9):664-667.
LI X, CHANG J T, LIU H J, et al. Seroepidemiological survey of the group A bovine rotavirus infection in China[J]. Chinese Journal of Preventive Veterinary Medicine, 2010, 32(9):664-667. (in Chinese)
[7] KOMOTO S, ADAH M I, IDE T, et al. Whole genomic analysis of human and bovine G8P
[1] rotavirus strains isolated in Nigeria provides evidence for direct bovine-to-human interspecies transmission[J]. Infect Genet Evol, 2016, 43:424-433.
[8] 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.
[9] KOMOTO S, TACHAROENMUANG R, GUNTAPONG R, et al. Reassortment of human and animal rotavirus gene segments in emerging DS-1-Like G1P
[8] rotavirus strains[J]. PLoS One, 2016, 11(2):e0148416.
[10] TACHAROENMUANG R, KOMOTO S, GUNTAPONG R, et al. Whole genomic analysis of an unusual human G6P
[14] rotavirus strain isolated from a child with diarrhea in Thailand:evidence for bovine-to-human interspecies transmission and reassortment events[J]. PLoS One, 2015, 10(9):e0139381.
[11] DENNIS F E, FUJII Y, HAGA K, et al. Identification of novel ghanaian G8P
[6] human-bovine reassortant rotavirus strain by next generation sequencing[J]. PLoS One, 2014, 9(6):e100699.
[12] MIJATOVIC-RUSTEMPASIC S, ESONA M D, WILLIAMS A L, et al. Sensitive and specific nested PCR assay for detection of rotavirus A in samples with a low viral load[J]. J Virol Methods, 2016, 236:41-46.
[13] 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)
[14] 李成元, 姚美玲, 王广伟, 等. 应用RT-PCR方法检测牛轮状病毒的研究[J]. 中国动物保健, 2015, 17(4):65-68.
LI C Y, YAO M L, WANG G W, et al. Study on the application of RT-PCR method for detection of bovine rotavirus[J]. China Animal Health, 2015, 17(4):65-68. (in Chinese)
[15] 周芳, 岳华, 张斌, 等. 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[J]. 畜牧兽医学报, 2016, 47(7):1465-1473.
ZHOU F, YUE H, ZHANG B, et al. 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. (in Chinese)
[16] KRISHNAN M, UGAZ V M, BURNA M A. PCR in a Rayleigh-Bénard convection cell[J]. Science, 2002, 298(5594):793.
[17] TSAI Y L, LIN Y C, CHOU P H, et al. Detection of white spot syndrome virus by polymerase chain reaction performed under insulated isothermal conditions[J]. J Virol Methods, 2012, 181(1):134-137.
[18] TSAI Y L, WANG H T T, CHANG H F G, et al. Development of TaqMan probe-based insulated isothermal PCR (iiPCR) for sensitive and specific on-site pathogen detection[J]. PLoS One, 2012, 7(9):e45278.
[19] BALASURIYA U B R, LEE P Y A, TIWARI A, et al. Rapid detection of equine influenza virus H3N8 subtype by insulated isothermal RT-PCR (iiRT-PCR) assay using the POCKIT? Nucleic Acid Analyzer[J]. J Virol Methods, 2014, 207:66-72.
[20] WILKES R P, TSAI Y L, LEE P Y, et al. Rapid and sensitive detection of canine distemper virus by one-tube reverse transcription-insulated isothermal polymerase chain reaction[J]. BMC Vet Res, 2014, 10:213.
[21] WILKES R P, LEE P Y A, TSAI Y L, et al. An insulated isothermal PCR method on a field-deployable device for rapid and sensitive detection of canine parvovirus type 2 at points of need[J]. J Virol Methods, 2015, 220:35-38.
[22] LUNG O, PASICK J, FISHER M, et al. Insulated isothermal reverse transcriptase PCR (iiRT-PCR) for Rapid and sensitive detection of classical swine fever virus[J]. Transbound Emerg Dis, 2016, 63(5):e395-e402.
[23] TSEN H Y, SHIH C M, TENG P H, et al. Detection of Salmonella in chicken meat by insulated isothermal PCR[J]. J Food Protect, 2013, 76(8):1322-1329.
[24] CHANG H F G, TSAI Y L, TSAI C F, et al. A thermally baffled device for highly stabilized convective PCR[J]. Biotechnol J, 2012, 7(5):662-666.
[25] 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 RotaTeq^® vaccine strains in stool samples[J]. Hum Vaccin Immunother, 2014, 10(3):767-777.
[26] PETER S G, GITAU G K, RICHARDS S, et al. Risk factors associated with Cryptosporidia, Eimeria, and diarrhea in smallholder dairy farms in Mukurwe-ini Sub-County, Nyeri County, Kenya[J]. Vet World, 2016, 9(8):811-819.
[27] CHEN X, ZHANG B, YUE H, et al. A novel astrovirus species in the gut of yaks with diarrhoea in the Qinghai-Tibetan plateau, 2013[J]. J Gen Virol, 2015, 96(12):3672-3280.
[28] HE H R, WU Y Q, HE N Y, et al. The latest progress of on-site pathogens detection techniques and instruments based on nucleic acid[J]. J Nanosci Nanotechnol, 2015, 15(9):6342-6356.
[29] GO Y Y, RAJAPAKSE R P V, KULARATNE S A M, et al. A pan-dengue virus reverse transcription-insulated isothermal PCR assay intended for point-of-need diagnosis of dengue virus infection by Use of the POCKIT Nucleic Acid Analyzer[J]. J Clin Microbiol, 2016, 54(6):1528-1535.

Development and Application of an Insulated Isothermal PCR (iiPCR) for On-site Detecting Bovine Rotavirus

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