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Development of real-time reverse transcriptase-polymerase chain reaction (rt RT- PCR) targeting four genes of peste des petits ruminants virus

Year 2015, Volume: 62 Issue: 4, 283 - 287, 01.12.2015

Ender Dinçer Aykut Özkul

https://doi.org/10.1501/Vetfak_0000002693

Abstract

In this study, one-step real-time quantitative reverse transcription PCR (qRT- PCR) was developed for the first time and evaluated for detection of peste des petits ruminants virus (PPRV) in field samples obtained from distinct geographical areas of Turkey. Primers and probes targeting four PPRV genes (namely L, M, N and P) were designed based on full genome sequence (AJ849636) of the local isolate (Tu00). The detection limits of the assays were found to be 7 copies/µL RNA for L, 6 copies/µL RNA for P, 10 copies/µL RNA for M and 700 copies/µL RNA for N, respectively. Besides, the detection ratio for PPRV in 45 field samples was 100% (45) for L, 88.9% (40) for M, 77.8% (35) for P and 22.3% (10) for N, respectively. In conclution, onestep real-time quantitative reverse transcription PCR (qRT- PCR) assay reported here for L gene design provides rapid, specific and sensitive detection of PPRV in tissue samples obtained from field cases

Keywords

Peste des petits ruminants Real-time qRT-PCR virus detection

References

  • Abera T, Thangavelu A, Chandran JDN, et al (2014): A SYBR Green I based real- time RT- PCR assay for specific detection and quantitation of Peste des petits ruminants virus. Vet Res, 10, 1-8.
  • Albayrak H, Gür S (2010): A serological investigation for Peste des petits ruminants infection in sheep, cattle and camels (Camelus dromedarious) in Aydın province, West Anatolia. Trop Anim Health Pro, 42, 151-153.
  • Balamurugan V, Sen A, Saravanan P, et al (2006): One- step multiplex RT-PCR assay for the detection of the peste des petits ruminants virus in clinical samples. Vet Res Commun, 30, 655-666.
  • Bao J, Li L, Wang Z, et al (2008): Development of one-step real-time RT-PCR assay for detection and quantitation of peste des petits ruminants virus. J Virol Methods, 148, 232-236.
  • Barrett T, Romeo CH, Baron MD, et al (1993): The molecular biology of rinderpest and peste des petits ruminants. Ann Med Vet, 137, 77-85.
  • Batten AC, Banyard CA, King PD, et al (2011): A real- time RT- PCR assay fort the specific detection of Peste des petits ruminants virus. J Virol Methods, 171, 401-404.
  • Couacy - Hymann E, Roger F, Hurard C, et al (2002): Rapid and Sensitive detection of the peste des petits ruminants virus by polymerase chain reaction assay. J Virol Methods, 100, 17- 25.
  • Diallo A, Libeau G, Couacy-Hymann E, et al (1995): Recent developments in the diagnosis of Rinderpest and Peste des petits ruminants. Vet Microbiol, 23, 307-317.
  • Grant JR, Banyard AC, Barrett T, et al (2009): Real- time RT-PCR assays for rapid and differential detection of dolphin and porpoise morbillivirus. J Virol Methods, 156, 117- 123.
  • Gulyaz V, Celen N, Ozkul A (2005): The isolation of PPR virus and pathogenicity and attenuation studies on
  • vero cell culture Pendik Vet Mikrobiyol Derg, 36, 1-2.
  • Gurcay M, Kizil O, Baydar E (2013): Peste Des Petits Ruminants (PPR) Virus infections in goats in the Eastern Anatolia of Turkey. Kafkas Univ Vet Fak Derg, 19, 93- 98.
  • Hughes GJ, Smith JS, Hanlon CA, et al (2004): Evaluation of a TaqMan PCR assay to detect rabies virus RNA: Influence of sequence variation and application to quantitation of viral loads. J Clin Microbiol, 42, 299-306.
  • Jimenez - Clavero MA, Aguero M, San Miguel E, et al (2006): High throughput detection of bluetongue virus by a new real-time fluorogenic reverse transcription - polymerase chain reaction: application on clinical samples from current Mediterranean outbreaks. J Vet Diagn Invest, 18, 7-17.
  • Kwiatek O, Minet C, Grillet C, et al (2007): Peste des petits ruminants (PPR) outbreak in Tajikistan. J Comp Pathol, 136, 111-119.
  • Kwiatek O, Keita D, Gil P, et al (2010): Quantitative one-step real-time RT- PCR for the fast detection of the four genotypes of PPRV. J Virol Methods, 165, 168-177.
  • Ozkul A, Akca Y, Alkan F, et al (2002): Prevalance, Distribution, and Host Range of Peste des petits ruminants virus, Turkey. Emerg Infect Dis, 8, 708-712.
  • Saliki JT, Cooper EJ, Gustavson JP (2002): Emerging morbillivirus infections of marine mammals: development of two diagnostic approaches. Ann NY Acad Sci, 969 51- 59.
  • Thomas B, Beard S, Jin L, et al (2007): Development and Evaluation of a Real- time PCR Assay for Rapid Identification and Semi-Quantitation of Measles. J Med Virol, 79, 1587-1592.

Küçük ruminant vebasının dört genini hedefleyen gerçek zamanlı reverz - transkriptaz polimerazzincir reaksiyonu (rtRT – PZR)’ nun geliştirilmesi

Year 2015, Volume: 62 Issue: 4, 283 - 287, 01.12.2015

Ender Dinçer Aykut Özkul

https://doi.org/10.1501/Vetfak_0000002693

Abstract

Bu çalışmada, tek basamaklı gerçek zamanlı kantitatif reverz transkripsiyon polimerize zincir reaksiyonu tekniği (qRTPCR) geliştirildi ve Türkiye’nin farklı coğrafik alanlarından elde edilen saha örneklerinde küçük ruminant vebası virusunun (PPRV) tespiti için karşılaştırıldı. Yerel PPRV (Tu00)’ nün dört genini (L, M, N ve P) hedefleyen primer ve problar ifade edilen virusun tüm genom dizini (AJ849636) temel alınarak tasarlandı. Çalışma kapsamında gerçekleştirilen tasarımlar ile her testin tespit sınırları, sırasıyla L geni için 7 kopya/µL RNA; P geni için 6 kopya/µL RNA; M geni için 10 kopya/µL RNA ve N geni için 700 kopya/µL RNA olarak bulundu. Bunun yanında, 45 saha örneği kullanılarak yapılan tanısal değerlendirmelerde ise virus genomunun tespit oranları sırasıyla L geni için %100 (45); M geni için %88.9 (40); P geni için %77.8 (35) ve N geni için %22.3 (10) olarak bulundu. Sonuç olarak, L geni dizaynı için bildirilen tek basamaklı gerçek zamanlı kantitatif reverz transkripsiyon polimerize zincir reaksiyonu deneyi (qRT-PCR) saha vakalarından elde edilen doku örneklerinde hızlı, spesifik ve hassas PPRV tespitini saplamıştır

Keywords

Küçük ruminant vebası Gerçek zamanlı RT-PZR virus tespiti

References

  • Abera T, Thangavelu A, Chandran JDN, et al (2014): A SYBR Green I based real- time RT- PCR assay for specific detection and quantitation of Peste des petits ruminants virus. Vet Res, 10, 1-8.
  • Albayrak H, Gür S (2010): A serological investigation for Peste des petits ruminants infection in sheep, cattle and camels (Camelus dromedarious) in Aydın province, West Anatolia. Trop Anim Health Pro, 42, 151-153.
  • Balamurugan V, Sen A, Saravanan P, et al (2006): One- step multiplex RT-PCR assay for the detection of the peste des petits ruminants virus in clinical samples. Vet Res Commun, 30, 655-666.
  • Bao J, Li L, Wang Z, et al (2008): Development of one-step real-time RT-PCR assay for detection and quantitation of peste des petits ruminants virus. J Virol Methods, 148, 232-236.
  • Barrett T, Romeo CH, Baron MD, et al (1993): The molecular biology of rinderpest and peste des petits ruminants. Ann Med Vet, 137, 77-85.
  • Batten AC, Banyard CA, King PD, et al (2011): A real- time RT- PCR assay fort the specific detection of Peste des petits ruminants virus. J Virol Methods, 171, 401-404.
  • Couacy - Hymann E, Roger F, Hurard C, et al (2002): Rapid and Sensitive detection of the peste des petits ruminants virus by polymerase chain reaction assay. J Virol Methods, 100, 17- 25.
  • Diallo A, Libeau G, Couacy-Hymann E, et al (1995): Recent developments in the diagnosis of Rinderpest and Peste des petits ruminants. Vet Microbiol, 23, 307-317.
  • Grant JR, Banyard AC, Barrett T, et al (2009): Real- time RT-PCR assays for rapid and differential detection of dolphin and porpoise morbillivirus. J Virol Methods, 156, 117- 123.
  • Gulyaz V, Celen N, Ozkul A (2005): The isolation of PPR virus and pathogenicity and attenuation studies on
  • vero cell culture Pendik Vet Mikrobiyol Derg, 36, 1-2.
  • Gurcay M, Kizil O, Baydar E (2013): Peste Des Petits Ruminants (PPR) Virus infections in goats in the Eastern Anatolia of Turkey. Kafkas Univ Vet Fak Derg, 19, 93- 98.
  • Hughes GJ, Smith JS, Hanlon CA, et al (2004): Evaluation of a TaqMan PCR assay to detect rabies virus RNA: Influence of sequence variation and application to quantitation of viral loads. J Clin Microbiol, 42, 299-306.
  • Jimenez - Clavero MA, Aguero M, San Miguel E, et al (2006): High throughput detection of bluetongue virus by a new real-time fluorogenic reverse transcription - polymerase chain reaction: application on clinical samples from current Mediterranean outbreaks. J Vet Diagn Invest, 18, 7-17.
  • Kwiatek O, Minet C, Grillet C, et al (2007): Peste des petits ruminants (PPR) outbreak in Tajikistan. J Comp Pathol, 136, 111-119.
  • Kwiatek O, Keita D, Gil P, et al (2010): Quantitative one-step real-time RT- PCR for the fast detection of the four genotypes of PPRV. J Virol Methods, 165, 168-177.
  • Ozkul A, Akca Y, Alkan F, et al (2002): Prevalance, Distribution, and Host Range of Peste des petits ruminants virus, Turkey. Emerg Infect Dis, 8, 708-712.
  • Saliki JT, Cooper EJ, Gustavson JP (2002): Emerging morbillivirus infections of marine mammals: development of two diagnostic approaches. Ann NY Acad Sci, 969 51- 59.
  • Thomas B, Beard S, Jin L, et al (2007): Development and Evaluation of a Real- time PCR Assay for Rapid Identification and Semi-Quantitation of Measles. J Med Virol, 79, 1587-1592.
There are 19 citations in total.

Details

Primary Language English
Subjects Veterinary Surgery
Other ID JA29PD43PU
Journal Section Research Article
Authors

Ender Dinçer

Aykut Özkul

Publication Date December 1, 2015
Published in Issue Year 2015Volume: 62 Issue: 4

Cite

APA Dinçer, E., & Özkul, A. (2015). Development of real-time reverse transcriptase-polymerase chain reaction (rt RT- PCR) targeting four genes of peste des petits ruminants virus. Ankara Üniversitesi Veteriner Fakültesi Dergisi, 62(4), 283-287. https://doi.org/10.1501/Vetfak_0000002693
AMA Dinçer E, Özkul A. Development of real-time reverse transcriptase-polymerase chain reaction (rt RT- PCR) targeting four genes of peste des petits ruminants virus. Ankara Univ Vet Fak Derg. December 2015;62(4):283-287. doi:10.1501/Vetfak_0000002693
Chicago Dinçer, Ender, and Aykut Özkul. “Development of Real-Time Reverse Transcriptase-Polymerase Chain Reaction (rt RT- PCR) Targeting Four Genes of Peste Des Petits Ruminants Virus”. Ankara Üniversitesi Veteriner Fakültesi Dergisi 62, no. 4 (December 2015): 283-87. https://doi.org/10.1501/Vetfak_0000002693.
EndNote Dinçer E, Özkul A (December 1, 2015) Development of real-time reverse transcriptase-polymerase chain reaction (rt RT- PCR) targeting four genes of peste des petits ruminants virus. Ankara Üniversitesi Veteriner Fakültesi Dergisi 62 4 283–287.
IEEE E. Dinçer and A. Özkul, “Development of real-time reverse transcriptase-polymerase chain reaction (rt RT- PCR) targeting four genes of peste des petits ruminants virus”, Ankara Univ Vet Fak Derg, vol. 62, no. 4, pp. 283–287, 2015, doi: 10.1501/Vetfak_0000002693.
ISNAD Dinçer, Ender - Özkul, Aykut. “Development of Real-Time Reverse Transcriptase-Polymerase Chain Reaction (rt RT- PCR) Targeting Four Genes of Peste Des Petits Ruminants Virus”. Ankara Üniversitesi Veteriner Fakültesi Dergisi 62/4 (December 2015), 283-287. https://doi.org/10.1501/Vetfak_0000002693.
JAMA Dinçer E, Özkul A. Development of real-time reverse transcriptase-polymerase chain reaction (rt RT- PCR) targeting four genes of peste des petits ruminants virus. Ankara Univ Vet Fak Derg. 2015;62:283–287.
MLA Dinçer, Ender and Aykut Özkul. “Development of Real-Time Reverse Transcriptase-Polymerase Chain Reaction (rt RT- PCR) Targeting Four Genes of Peste Des Petits Ruminants Virus”. Ankara Üniversitesi Veteriner Fakültesi Dergisi, vol. 62, no. 4, 2015, pp. 283-7, doi:10.1501/Vetfak_0000002693.
Vancouver Dinçer E, Özkul A. Development of real-time reverse transcriptase-polymerase chain reaction (rt RT- PCR) targeting four genes of peste des petits ruminants virus. Ankara Univ Vet Fak Derg. 2015;62(4):283-7.