A rapid and sensitive one step-SYBR green based semi quantitative real time RT-PCR for the detection of peste des petits ruminants virus in the clinical samples.
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Title |
A rapid and sensitive one step-SYBR green based semi quantitative real time RT-PCR for the detection of peste des petits ruminants virus in the clinical samples.
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Creator |
Balamurugan V, Sen A, Venkatesan G, Yadav V, Bhanot V, Bhanuprakash V, Singh RK.
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Subject |
SYBR Green, RT PCR, Detection, PPRV, Clinical samples
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Description |
A sensitive and rapid single step real time (rt) RT-PCR was standardized using one-step Brilliant SYBR Green kit® for detection and semi-quantitation of peste des petitis ruminants virus (PPRV) using the virus RNA and matrix (M) protein gene-specific primers and compared with established conventional RT-PCR and TaqMan RT-PCR. The assay amplifies a 124 bp fragment of the PPRV M gene with T(m) of 78.28 to 78.50. The assay was linear within a range of 50 ng to 0.5 fg total virus RNA with a detection limit (sensitivity) of 0.5 fg. Based on the serial dilution of the live-attenuated PPR vaccine virus, the detection limit was ~0.0001 cell culture infectious dose 50% units (TCID(50)). Additionally, swab materials spiked with known titre of vaccine virus were equally well detected in the assay. The standardized rt RT-PCR was easily employed for the detection of PPRV nucleic acid directly in the field and experimental clinical samples. The assay detected the PPRV nucleic acid as early as 3 day post infection (dpi) and up to 20 dpi in swab materials from the experimental samples. The assay was rapid and more sensitive than TaqMan and conventional RT-PCR in the detection of PPRV nucleic acid from the PPR suspected clinical samples of sheep and goats. Therefore, the established, simplified SYBR green rt RT-PCR is an alternative test to the already existing various diagnostic assays and could be useful for rapid clinical diagnosis with advantage in reducing risk of contamination.
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Date |
2023-05-12T10:28:35Z
2023-05-12T10:28:35Z 2012-02-01 |
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Type |
Research Paper
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Identifier |
Not Available
http://krishi.icar.gov.in/jspui/handle/123456789/77240 |
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Language |
English
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Publisher |
Elsevier
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