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Home / Equine Research Bank / Front Microbiol / Establishment of a reverse transcription real-time quantitat...
Frontiers in microbiology2022; 13; 1009610; doi: 10.3389/fmicb.2022.1009610

Establishment of a reverse transcription real-time quantitative PCR method for Getah virus detection and its application for epidemiological investigation in Shandong, China.

Abstract: Getah virus (GETV) is a mosquito-borne, single-stranded, positive-sense RNA virus belonging to the genus of the family . Natural infections of GETV have been identified in a variety of vertebrate species, with pathogenicity mainly in swine, horses, bovines, and foxes. The increasing spectrum of infection and the characteristic causing abortions in pregnant animals pose a serious threat to public health and the livestock economy. Therefore, there is an urgent need to establish a method that can be used for epidemiological investigation in multiple animals. In this study, a real-time reverse transcription fluorescent quantitative PCR (RT-qPCR) method combined with plaque assay was established for GETV with specific primers designed for the highly conserved region of GETV gene. The results showed that after optimizing the condition of RT-qPCR reaction, the minimum detection limit of the assay established in this study was 7.73 PFU/mL, and there was a good linear relationship between viral load and value with a correlation coefficient ( ) of 0.998. Moreover, the method has good specificity, sensitivity, and repeatability. The established RT-qPCR is 100-fold more sensitive than the conventional RT-PCR. The best cutoff value for the method was determined to be 37.59 by receiver operating characteristic (ROC) curve analysis. The area under the curve (AUC) was 0.956. Meanwhile, we collected 2,847 serum specimens from swine, horses, bovines, sheep, and 17,080 mosquito specimens in Shandong Province in 2022. The positive detection rates by RT-qPCR were 1%, 1%, 0.2%, 0%, and 3%, respectively. In conclusion, the method was used for epidemiological investigation, which has extensive application prospects.
Copyright © 2022 Cao, Qiu, Shi, Ha, Zhang, Xie, Wang, Zhu, Zhao, Zhao, Jin and Lu.
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Publication Date: 2022-09-23 PubMed ID: 36212868PubMed Central: PMC9538719DOI: 10.3389/fmicb.2022.1009610Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The study details the establishment of a real-time reverse transcription quantitative PCR (shortened to RT-qPCR) method for detecting the Getah virus, a mosquito-borne RNA virus known to cause illnesses in several species of animals. The researchers tested the method on numerous serum and mosquito samples collected in the Shandong Province, China, and concluded that the method can be very useful for epidemiological investigations.

Development of the RT-qPCR method for Getah virus detection

  • The researchers set out to design a RT-qPCR method for Getah virus (GETV) detection with the use of specific primers for the highly conserved region of the GETV NSP1 gene. This method was created in response to the virus’s increasing spectrum of infection and harmful effects, particularly in pregnant animals.
  • GETV is known to infect a range of vertebrate species and causes severe health complications, including the potential to cause abortion in pregnant animals, thus posing a significant risk not only to public health but also to the livestock economy.

Features and accuracy of the RT-qPCR method

  • The RT-qPCR method showed high accuracy, having a minimum detection limit established at 7.73 PFU/mL. In simpler terms, this refers to how sensitive the test is in detecting the smallest amount of virus present in test samples.
  • The correlation coefficient of the method (shown as R) was 0.998, meaning there was a very high degree of accuracy between viral load and the ‘Ct’ value obtained during testing.
  • The method showed strong specificity, sensitivity, and repeatability, making it a reliable way to detect GETV.
  • The researchers also noted that their RT-qPCR method was 100-fold more sensitive than the conventional RT-PCR method.
  • The best cutoff value for the method was determined to be 37.59 by receiver operating characteristic (ROC) curve analysis. The area under the curve (AUC), another measure of test accuracy, was 0.956–another indication of the test’s high accuracy.

Application of the RT-qPCR method for epidemiological investigation

  • The developed method was used to test on 2,847 serum specimens from swine, horses, bovines, sheep, and 17,080 mosquito specimens collected in 2022 in the Shandong province of China.
  • Results showed varying positive detection rates for different species: 1% for swine and horses, 0.2% for bovines, 0% for sheep, and 3% for the mosquito specimens.
  • The researchers concluded that the RT-qPCR method they developed holds potential for broader application in epidemiological investigations of GETV.

Cite This Article

APA
Cao X, Qiu X, Shi N, Ha Z, Zhang H, Xie Y, Wang P, Zhu X, Zhao W, Zhao G, Jin N, Lu H. (2022). Establishment of a reverse transcription real-time quantitative PCR method for Getah virus detection and its application for epidemiological investigation in Shandong, China. Front Microbiol, 13, 1009610. https://doi.org/10.3389/fmicb.2022.1009610

Publication

Frontiers in microbiology
ISSN: 1664-302X
NlmUniqueID: 101548977
Country: Switzerland
Language: English
Volume: 13
Pages: 1009610
PII: 1009610

Researcher Affiliations

Cao, Xinyu
  • Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Changchun, Jilin, China.
  • College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China.
Qiu, Xiangshu
  • Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Changchun, Jilin, China.
  • College of Animal Sciences, Institute of Preventive Veterinary Medicine, Zhejiang University, Hangzhou, Zhejiang, China.
Shi, Ning
  • Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Changchun, Jilin, China.
  • Key Laboratory of Zoonoses Research, Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, Changchun, China.
Ha, Zhuo
  • Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Changchun, Jilin, China.
Zhang, He
  • Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Changchun, Jilin, China.
Xie, Yubiao
  • Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Changchun, Jilin, China.
Wang, Peng
  • Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Changchun, Jilin, China.
Zhu, Xiangyu
  • Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Changchun, Jilin, China.
Zhao, Wenxin
  • Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Changchun, Jilin, China.
Zhao, Guanyu
  • Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Changchun, Jilin, China.
  • Key Laboratory of Zoonoses Research, Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, Changchun, China.
Jin, Ningyi
  • Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Changchun, Jilin, China.
  • College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China.
  • College of Animal Sciences, Institute of Preventive Veterinary Medicine, Zhejiang University, Hangzhou, Zhejiang, China.
  • Key Laboratory of Zoonoses Research, Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, Changchun, China.
Lu, Huijun
  • Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Changchun, Jilin, China.
  • College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

References

This article includes 39 references
  1. Acevedo AM, Perera CL, Vega A, Ríos L, Coronado L, Relova D, Frías MT, Ganges L, Núñez JI, Pérez LJ. A duplex SYBR Green I-based real-time RT-PCR assay for the simultaneous detection and differentiation of Massachusetts and non-Massachusetts serotypes of infectious bronchitis virus.. Mol Cell Probes 2013 Oct-Dec;27(5-6):184-92.
    doi: 10.1016/j.mcp.2013.06.001pubmed: 23810983google scholar: lookup
  2. Bae HG, Nitsche A, Teichmann A, Biel SS, Niedrig M. Detection of yellow fever virus: a comparison of quantitative real-time PCR and plaque assay.. J Virol Methods 2003 Jun 30;110(2):185-91.
    doi: 10.1016/s0166-0934(03)00129-0pubmed: 12798247google scholar: lookup
  3. Bannai H, Nemoto M, Ochi A, Kikuchi T, Kobayashi M, Tsujimura K, Yamanaka T, Kondo T. Epizootiological Investigation of Getah Virus Infection among Racehorses in Japan in 2014.. J Clin Microbiol 2015 Jul;53(7):2286-91.
    doi: 10.1128/jcm.00550-15pmc: PMC4473224pubmed: 25972425google scholar: lookup
  4. Dong D, Fu SH, Wang LH, Lv Z, Li TY, Liang GD. Simultaneous detection of three arboviruses using a triplex RT-PCR: enzyme hybridization assay.. Virol Sin 2012 Jun;27(3):179-86.
    doi: 10.1007/s12250-012-3246-9pmc: PMC3724924pubmed: 22684472google scholar: lookup
  5. Fumagalli MJ, de Souza WM, de Castro-Jorge LA, de Carvalho RVH, Castro ÍA, de Almeida LGN, Consonni SR, Zamboni DS, Figueiredo LTM. Chikungunya Virus Exposure Partially Cross-Protects against Mayaro Virus Infection in Mice.. J Virol 2021 Nov 9;95(23):e0112221.
    doi: 10.1128/jvi.01122-21pmc: PMC8577356pubmed: 34549980google scholar: lookup
  6. Gao S, Du J, Tian Z, Niu Q, Huang D, Wang J, Luo J, Liu G, Yin H. A SYBR green I-based quantitative RT-PCR assay for bovine ephemeral fever virus and its utility for evaluating viral kinetics in cattle.. J Vet Diagn Invest 2020 Jan;32(1):44-50.
    doi: 10.1177/1040638719895460pmc: PMC7003230pubmed: 31845623google scholar: lookup
  7. Garcia S, Crance JM, Billecocq A, Peinnequin A, Jouan A, Bouloy M, Garin D. Quantitative real-time PCR detection of Rift Valley fever virus and its application to evaluation of antiviral compounds.. J Clin Microbiol 2001 Dec;39(12):4456-61.
    doi: 10.1128/jcm.39.12.4456-4461.2001pmc: PMC88565pubmed: 11724861google scholar: lookup
  8. Guillaume V, Lefeuvre A, Faure C, Marianneau P, Buckland R, Lam SK, Wild TF, Deubel V. Specific detection of Nipah virus using real-time RT-PCR (TaqMan).. J Virol Methods 2004 Sep 15;120(2):229-37.
    doi: 10.1016/j.jviromet.2004.05.018pubmed: 15288966google scholar: lookup
  9. Hughes G. Youden's index and the weight of evidence.. Methods Inf Med 2015;54(2):198-9.
    doi: 10.3414/ME14-04-0003pubmed: 25658987google scholar: lookup
  10. Houng HH, Hritz D, Kanesa-thasan N. Quantitative detection of dengue 2 virus using fluorogenic RT-PCR based on 3'-noncoding sequence.. J Virol Methods 2000 Apr;86(1):1-11.
    doi: 10.1016/s0166-0934(99)00166-4pubmed: 10713370google scholar: lookup
  11. Hu T, Zheng Y, Zhang Y, Li G, Qiu W, Yu J, Cui Q, Wang Y, Zhang C, Zhou X, Feng Z, Zhou W, Fan Q, Zhang F. Identification of a novel Getah virus by Virus-Discovery-cDNA random amplified polymorphic DNA (RAPD).. BMC Microbiol 2012 Dec 27;12:305.
    doi: 10.1186/1471-2180-12-305pmc: PMC3547691pubmed: 23268691google scholar: lookup
  12. King DP, Ferris NP, Shaw AE, Reid SM, Hutchings GH, Giuffre AC, Robida JM, Callahan JD, Nelson WM, Beckham TR. Detection of foot-and-mouth disease virus: comparative diagnostic sensitivity of two independent real-time reverse transcription-polymerase chain reaction assays.. J Vet Diagn Invest 2006 Jan;18(1):93-7.
    doi: 10.1177/104063870601800114pubmed: 16566264google scholar: lookup
  13. Kuwata R, Shimoda H, Phichitraslip T, Prasertsincharoen N, Noguchi K, Yonemitsu K, Minami S, Supriyono, Tran NTB, Takano A, Suzuki K, Nemoto M, Bannai H, Yokoyama M, Takeda T, Jittapalapong S, Rerkamnuaychoke W, Maeda K. Getah virus epizootic among wild boars in Japan around 2012.. Arch Virol 2018 Oct;163(10):2817-2821.
    doi: 10.1007/s00705-018-3897-4pubmed: 29876783google scholar: lookup
  14. La Scola B, Le Bideau M, Andreani J, Hoang VT, Grimaldier C, Colson P, Gautret P, Raoult D. Viral RNA load as determined by cell culture as a management tool for discharge of SARS-CoV-2 patients from infectious disease wards.. Eur J Clin Microbiol Infect Dis 2020 Jun;39(6):1059-1061.
    doi: 10.1007/s10096-020-03913-9pmc: PMC7185831pubmed: 32342252google scholar: lookup
  15. Leifer I, Everett H, Hoffmann B, Sosan O, Crooke H, Beer M, Blome S. Escape of classical swine fever C-strain vaccine virus from detection by C-strain specific real-time RT-PCR caused by a point mutation in the primer-binding site.. J Virol Methods 2010 Jun;166(1-2):98-100.
    doi: 10.1016/j.jviromet.2010.03.004pubmed: 20332004google scholar: lookup
  16. Liu H, Li LX, Bu YP, Liu Y, Sun XT, Shi N, Deng XY, Lu RG, Hu B, Jin NY, Yan XJ. Rapid Visual Detection of Getah Virus Using a Loop-Mediated Isothermal Amplification Method.. Vector Borne Zoonotic Dis 2019 Oct;19(10):741-746.
    doi: 10.1089/vbz.2018.2434pubmed: 30964395google scholar: lookup
  17. Lu G, Chen R, Shao R, Dong N, Liu W, Li S. Getah virus: An increasing threat in China.. J Infect 2020 Mar;80(3):350-371.
    doi: 10.1016/j.jinf.2019.11.016pubmed: 31790706google scholar: lookup
  18. Lu G, Ou J, Ji J, Ren Z, Hu X, Wang C, Li S. Emergence of Getah Virus Infection in Horse With Fever in China, 2018.. Front Microbiol 2019;10:1416.
    doi: 10.3389/fmicb.2019.01416pmc: PMC6596439pubmed: 31281304google scholar: lookup
  19. Marchette NJ, Rudnick A, Garcia R, MacVean DW. Alphaviruses in Peninusular Malaysia: I. Virus isolations and animal serology.. Southeast Asian J Trop Med Public Health 1978 Sep;9(3):317-29.
    pubmed: 34888
  20. McMillen CM, Arora N, Boyles DA, Albe JR, Kujawa MR, Bonadio JF, Coyne CB, Hartman AL. Rift Valley fever virus induces fetal demise in Sprague-Dawley rats through direct placental infection.. Sci Adv 2018 Dec;4(12):eaa頒.
    doi: 10.1126/sciadv.aa頒pmc: PMC6281433pubmed: 30525107google scholar: lookup
  21. Morita K, Igarashi A. Oligonucleotide fingerprint analysis of strains of Getah virus isolated in Japan and Malaysia.. J Gen Virol 1984 Nov;65 ( Pt 11):1899-908.
    doi: 10.1099/0022-1317-65-11-1899pubmed: 6094708google scholar: lookup
  22. Murphy FA, Fauquet CM, Bishop D, Ghabrial SA, Summers MD. Virus taxonomy: classification and nomenclature of viruses. Encyclopedia Virol 140, 9–23.
    doi: 10.1007/BF01309873google scholar: lookup
  23. Nemoto M, Bannai H, Tsujimura K, Kobayashi M, Kikuchi T, Yamanaka T, Kondo T. Getah Virus Infection among Racehorses, Japan, 2014.. Emerg Infect Dis 2015 May;21(5):883-5.
    doi: 10.3201/eid2105.141975pmc: PMC4412242pubmed: 25898181google scholar: lookup
  24. Ogawa H, Taira O, Hirai T, Takeuchi H, Nagao A, Ishikawa Y, Tuchiya K, Nunoya T, Ueda S. Multiplex PCR and multiplex RT-PCR for inclusive detection of major swine DNA and RNA viruses in pigs with multiple infections.. J Virol Methods 2009 Sep;160(1-2):210-4.
    doi: 10.1016/j.jviromet.2009.05.010pubmed: 19467264google scholar: lookup
  25. Powers AM, Brault AC, Shirako Y, Strauss EG, Kang W, Strauss JH, Weaver SC. Evolutionary relationships and systematics of the alphaviruses.. J Virol 2001 Nov;75(21):10118-31.
    doi: 10.1128/jvi.75.21.10118-10131.2001pmc: PMC114586pubmed: 11581380google scholar: lookup
  26. Ren T, Mo Q, Wang Y, Wang H, Nong Z, Wang J, Niu C, Liu C, Chen Y, Ouyang K, Huang W, Wei Z. Emergence and Phylogenetic Analysis of a Getah Virus Isolated in Southern China.. Front Vet Sci 2020;7:552517.
    doi: 10.3389/fvets.2020.552517pmc: PMC7744783pubmed: 33344520google scholar: lookup
  27. Sam SS, Teoh BT, Chee CM, Mohamed-Romai-Noor NA, Abd-Jamil J, Loong SK, Khor CS, Tan KK, AbuBakar S. A quantitative reverse transcription-polymerase chain reaction for detection of Getah virus.. Sci Rep 2018 Dec 5;8(1):17632.
    doi: 10.1038/s41598-018-36043-6pmc: PMC6281642pubmed: 30518924google scholar: lookup
  28. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method.. Nat Protoc 2008;3(6):1101-8.
    doi: 10.1038/nprot.2008.73pubmed: 18546601google scholar: lookup
  29. Shi N, Li LX, Lu RG, Yan XJ, Liu H. Highly Pathogenic Swine Getah Virus in Blue Foxes, Eastern China, 2017.. Emerg Infect Dis 2019 Jun;25(6):1252-1254.
    doi: 10.3201/eid2506.181983pmc: PMC6537705pubmed: 31107236google scholar: lookup
  30. Shi N, Liu H, Li LX, Hu B, Zhang L, Zhao CF, Deng XY, Li XT, Xue XH, Bai X, Zhang HL, Lu RG, Lian SZ, Wang Y, Yan MH, Yan XJ. Development of a TaqMan probe-based quantitative reverse transcription PCR assay for detection of Getah virus RNA.. Arch Virol 2018 Oct;163(10):2877-2881.
    doi: 10.1007/s00705-018-3927-2pubmed: 29987379google scholar: lookup
  31. Shi N, Zhu X, Qiu X, Cao X, Jiang Z, Lu H, Jin N. Origin, genetic diversity, adaptive evolution and transmission dynamics of Getah virus.. Transbound Emerg Dis 2022 Jul;69(4):e1037-e1050.
    doi: 10.1111/tbed.14395pubmed: 34812572google scholar: lookup
  32. Shibata I, Hatano Y, Nishimura M, Suzuki G, Inaba Y. Isolation of Getah virus from dead fetuses extracted from a naturally infected sow in Japan.. Vet Microbiol 1991 May;27(3-4):385-91.
    doi: 10.1016/0378-1135(91)90162-9pubmed: 1652864google scholar: lookup
  33. Shrestha NK, Marco Canosa F, Nowacki AS, Procop GW, Vogel S, Fraser TG, Erzurum SC, Terpeluk P, Gordon SM. Distribution of Transmission Potential During Nonsevere COVID-19 Illness.. Clin Infect Dis 2020 Dec 31;71(11):2927-2932.
    doi: 10.1093/cid/ciaa886pmc: PMC7337652pubmed: 32594116google scholar: lookup
  34. Tajima S, Kotaki A, Yagasaki K, Taniwaki T, Moi ML, Nakayama E, Saijo M, Kurane I, Takasaki T. Identification and amplification of Japanese encephalitis virus and Getah virus propagated from a single porcine serum sample: a case of coinfection.. Arch Virol 2014 Nov;159(11):2969-75.
    doi: 10.1007/s00705-014-2152-xpubmed: 24986716google scholar: lookup
  35. Tam S, Clavijo A, Engelhard EK, Thurmond MC. Fluorescence-based multiplex real-time RT-PCR arrays for the detection and serotype determination of foot-and-mouth disease virus.. J Virol Methods 2009 Nov;161(2):183-91.
    doi: 10.1016/j.jviromet.2009.04.033pubmed: 19427333google scholar: lookup
  36. Xia YH, Shi ZC, Wang XW, Li YT, Wang Z, Chang HT, Liu HY, Chen L, Wang CQ, Yang X. Development and application of SYBR Green Ⅰ real-time quantitative reverse transcription PCR assay for detection of swine Getah virus.. Mol Cell Probes 2021 Jun;57:101730.
    doi: 10.1016/j.mcp.2021.101730pubmed: 33848593google scholar: lookup
  37. Xing C, Jiang J, Lu Z, Mi S, He B, Tu C, Liu X, Gong W. Isolation and characterization of Getah virus from pigs in Guangdong province of China.. Transbound Emerg Dis 2020 Apr 10;.
    doi: 10.1111/tbed.13567pubmed: 32277601google scholar: lookup
  38. Zhang Y, Li Y, Guan Z, Yang Y, Zhang J, Sun Q, Li B, Qiu Y, Liu K, Shao D, Ma Z, Wei J, Li P. Rapid Differential Detection of Japanese Encephalitis Virus and Getah Virus in Pigs or Mosquitos by a Duplex TaqMan Real-Time RT-PCR Assay.. Front Vet Sci 2022;9:839443.
    doi: 10.3389/fvets.2022.839443pmc: PMC9023051pubmed: 35464361google scholar: lookup
  39. Zhu Z, Fan H, Qi X, Qi Y, Shi Z, Wang H, Cui L, Zhou M. Development and evaluation of a SYBR green-based real time RT-PCR assay for detection of the emerging avian influenza A (H7N9) virus.. PLoS One 2013;8(11):e80028.
    doi: 10.1371/journal.pone.0080028pmc: PMC3835827pubmed: 24278234google scholar: lookup

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