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Single-tube nested reverse transcription-polymerase chain reaction for simultaneous detection of genotyping of porcine reproductive and respiratory syndrome virus without DNA carryover contamination
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  • Journal title : Korean Journal of Veterinary Service
  • Volume 39, Issue 2,  2016, pp.107-116
  • Publisher : The Korean Society of Veterinary Service
  • DOI : 10.7853/kjvs.2016.39.2.107
 Title & Authors
Single-tube nested reverse transcription-polymerase chain reaction for simultaneous detection of genotyping of porcine reproductive and respiratory syndrome virus without DNA carryover contamination
Jeong, Pil-Soo; Park, Su-Jin; Kim, Eun-Mi; Park, Ji-Young; Park, Yu-Ri; Kang, Dae-Young; Cha, Hyun-Ouk; Lee, Kyoung-Ki; Kim, Seong-Hee; Park, Choi-Kyu;
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 Abstract
In the study, we developed and evaluated a uracil N-glycosylase (UNG)-supplemented single-tube nested reverse transcription-polymerase chain reaction (UsnRT-PCR) assay that can carried out first-round RT-PCR and second-round nested PCR in a reaction tube without reaction tube opening and can simultaneously detect EU- and NA-PRRSV. The UsnRT-PCR confirmed to have a preventing ability of mis-amplification by contamination of pre-amplified PRRSV DNA from previous UsnRT-PCR. Primer specificities were evaluated with RNAs extracted from 8 viral strains and our results revealed that the primers had a high specificity for both genotypes of PRRSV. The sensitivity of the UsnRT-PCR was 0.1 /0.1 mL for EU- or NA-PRRSV, respectively, which is comparable to that of previously reported real time RT-PCR (RRT-PCR). Clinical evaluation on 110 field samples (60 sera and 50 lung tissues) by the UsnRT-PCR and the RRT-PCR showed that detection rates of the UsnRT-PCR was 70% (77/110), and was relatively higher than that of the RRT-PCR (69.1%, 76/110). The percent positive or negative agreement of the UsnRT-PCR compared to RRT-PCR was 96.1% (73/76) or 90.9% (30/33), showing that the test results of both assays may be different for some clinical samples. Therefore, it is recommend that diagnostic laboratory workers use the two diagnostic assays for the correct diagnosis for the relevant samples in the swine disease diagnostic laboratories. In conclusion, the UsnRT-PCR assay can be applied for the rapid, and reliable diagnosis of PRRSV without concerns about preamplified DNA carryover contamination that can occurred in PCR process in the swine disease diagnostic laboratories.
 Keywords
Porcine reproductive and respiratory syndrome virus (PRRSV);Single-tube nested reverse transcription-polymerase chain reaction (snRT-PCR);Uracil DNA glycosylase (UNG);
 Language
Korean
 Cited by
 References
1.
Abath FGC, Melo FL, Werkhauser RP, Montenegro L, Montenegro R and Schindler HC. 2002. Single-tube nested PCR using immobilizing internal primers. Biotechniques 33: 1210-1214.

2.
Albina E. 1997. Epidemiology of porcine reproductive and respiratory syndrome (PRRS): an overview. Vet Microbiol 55: 309-316. crossref(new window)

3.
Aslanzadeh J. 2004. Preventing PCR Amplification Carryover Contamination in a Clinical Laboratory. Annals Clin Lab Sci 34: 389-396.

4.
Christopher-Hennings J, Nelson EA, Nelson JK, Hines RJ, Swenson SL, Hill HT, Zimmerman JJ, Katz JB, Yaeger MJ, Chase CC. 1995. Detection of porcine reproductive and respiratory syndrome virus in boar semen by PCR. J Clin Microbiol 33: 1730-1734.

5.
Corzo CA, Mondaca E, Wayne S, Torremorell M, Dee S, Davies P, Morrison RB. 2010. Control and elimination of porcine reproductive and respiratory syndrome virus. Virus Res 154: 185-192. crossref(new window)

6.
Gilbert SA, Larochelle R, Magar R, Cho HJ, Deregtet D. 1997. Typing of porcine reproductive and respiratory syndrome viruses by a multiplex PCR assay. J Clin Microbiol 35: 264-267.

7.
Haff LA. 1994. Improved quantitative PCR using nested PCR. PCR methods applic 3: 332-337. crossref(new window)

8.
Indik S, Schmoll F, Sipos W, Klein D. 2005. Genetic variability of PRRS virus in Austria: consequences for molecular diagnostics and viral quantification. Vet Microbiol 107: 171-178. crossref(new window)

9.
Keffaber KK, 1989. Reproductive failure of unknown etiology. Am Assoc Swine Pract Newslett 1: 1-9.

10.
Kim EM, Park CK. 2014. UNG-based direct polymerase chain reaction (udPCR) for the detection of porcine circovirus 2 (PCV2). Korean J Vet Serv 37: 253-261. crossref(new window)

11.
Kim HS, Kwang J, Yoon IJ, Joo HS, Frey ML. 1993. Enhanced replication of porcine reproductive and respiratory syndrome (PRRS) virus in a homogeneous subpopulation of MA-104 cell line. Arch Virol 133: 477-483. crossref(new window)

12.
Kim SH, Roh S, Choi EJ, Lee C, Lee CH, Lee KH, Lee KK, Song YK, Lee OS, Park CK, 2010. A molecular analysis of European porcine reproductive and respiratory syndrome virus isolated in South Korea. Vet Microbiol 143: 394-400. crossref(new window)

13.
Kleiboeker SB, Schommer SK, Lee SM, Watkins S, Chittick W, Polson D. 2005. Simultaneous detection of North American and European porcine reproductive and respiratory syndrome virus using real-time quantitative reverse transcriptase-PCR. J Vet Diagn Invest 17: 165-170. crossref(new window)

14.
Kono Y, Kanno T, Shimizu M, Yamada S, Ohashi S, Nakamine M, Shirai J. 1996. Nested PCR for detection and typing of porcine reproductive and respiratory syndrome (PRRS) virus in pigs. J Vet Med Sci 58: 941-946. crossref(new window)

15.
Kwok S, Higuchi R. 1989. Avoiding false positives with PCR. Nature (London) 339: 237-238. crossref(new window)

16.
Lager KM, Mengeling WL, Brockmeier SL. 1997. Duration of homologous porcine reproductive and respiratory syndrome virus immunity in pregnant swine. Vet Microbiol 58: 127-133. crossref(new window)

17.
Longo MC, Berninger MS, Hartley JL. 1990. Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene 93: 125-128. crossref(new window)

18.
Murtaugh MP, Stadejek T, Abrahante JE, Lam TTY, Leung FCC. 2010. The ever-expanding diversity of porcine reproductive and respiratory syndrome virus. Virus Res 154: 18-30. crossref(new window)

19.
Nuemann EJ, Kliebenstein JB, Johnson CD, Mabry JW, Bush EJ, Seitzinger AH, Green AL, Zimmerman JJ. 2005. Assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the United States. J Am Vet Med Assoc 227: 385-392. crossref(new window)

20.
Pang J, Modlin J, Yolken R. 1992. Use of modified nucleotides and uracil-DNA glycosylase (UNG) for the control of contamination in the PCR-based amplification of RNA. Mol Cel Probes 6: 251-256. crossref(new window)

21.
Persing DH. 1990. Polymerase chain reaction: trenches to benches. J Clin Microbiol 29: 1281-1285.

22.
Poddar SK, Sawyer MH and Connor JD. 1997. Optimized PCR amplification of influenza A virus RNA using Tth DNA polymerase, incorporating uracil N glycosylase (UNG) in a single tube reaction. J Clin Lab Anal 11: 323-327. crossref(new window)

23.
Reed LJ, Muench H. 1938. A simple method of estimating fifty percent end points. Am J Hyg 27: 709-716.

24.
Rolfs A, Scholler I, Finckh U, Rolfs JW. 1992. General applications of PCR. In: Rolfs, A. (ed) PCR: clinical diagnostics and research. Springer-Verlag, Berlin, pp 34-50.

25.
Shi M, Lam TTY, Hon CC, Hui RKH, Faaberg KS, Wennblom T, Murtaugh MP, Stadejek T, Leung FCC. 2010. Molecular epidemiology of PRRSV: A phylogenetic perspective. Virus Res 154: 7-17. crossref(new window)

26.
Shin J, Bautista EM, Kang YB, Molitor TW. 1998. Quantitation of porcine reproductive and respiratory syndrome virus RNA in semen by single-tube reverse transcription-nested polymerase chain reaction. J Virol Methods 72: 67-69. crossref(new window)

27.
Thanawongnuwech R, Suradhat S. 2010. Taming PRRSV: revisiting the control strategies and vaccine design. Virus Res 154: 133-140. crossref(new window)

28.
Wensvoort G, Tepstra C, Pol JMA, ter Laak EA, Bloemraad M, de Kluyver EP, Kragten C, van Buiten L, den Besten A, Wagenaar F, Broekhuijsen JM, Moonen PLJM, Zestra T, de Boer EA, Tibben HJ, de Jong MF, van Veld P, Groenland GJR, van Gennep JA, Voetsd MTH, Verheijdene JHM, Braamskamp J. 1991. Mystery swine disease in The Netherlands: the isolation of Lelystad virus. Vet Q 13: 121-130. crossref(new window)