High throughput detection of retrovirus-associated reverse transcriptase using an improved fluorescent product enhanced reverse transcriptase assay and its comparison to conventional detection methods

被引：68

作者：

Lovatt, A ^{[1
]}

Black, J ^{[1
]}

Galbraith, D ^{[1
]}

Doherty, I ^{[1
]}

Moran, MW ^{[1
]}

Shepherd, AJ ^{[1
]}

Griffen, A ^{[1
]}

Bailey, A ^{[1
]}

Wilson, N ^{[1
]}

Smith, KT ^{[1
]}

机构：

[1] Q One Biotech Ltd, Glasgow G20 0XA, Lanark, Scotland

来源：

JOURNAL OF VIROLOGICAL METHODS | 1999年 / 82卷 / 02期

关键词：

retrovirus; product enhanced reverse transcriptase assay; PBRT; TaqMan PCR; calf thymus DNA; DNA polymerase; xenotransplantation;

D O I：

10.1016/S0166-0934(99)00111-1

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

The development and application of a novel, sensitive TaqMan fluorescent probe-based product enhanced RT test (F-PERT) for the detection of retrovirus are described. The assay allows discrimination between the amplification signals generated by genuine positive signals that result from retroviral RT activity and the RT-like activity from DNA polymerases. The RT-like activity from DNA polymerases was suppressed by the addition of activated calf-thymus DNA with no reduction in the RT activity. A linear relationship between threshold cycle (C-T) and the number of virus particles was demonstrated, allowing quantification of retroviruses in unknown samples. The F-PERT assay was able to detect a wide range of retroviral RT activities, including that from porcine endogenous retrovirus (PoERV), murine leukaemia virus (MLV), simian foamy virus (SFV), simian immunodeficiency virus (SIVmac) and squirrel monkey retrovirus (SMRV). The detection limit of SMRV, MLV and PoERV was approximately 100 virion particles and the test was able to detect at least 10(2) molecules of purified RT enzyme. RT activity was not detected in cellular lysates and supernatants from MRC-5, BT, VERO, or Raji cells, whereas RT activity was detected in C127I, Mus dunni. K-Balb, BHK-21, CHO-K1, SP2/0-Ag14 and NSO cell supernatants. RT activity was also detected in the Spodoptera cell line Sf9. (C) 1999 Elsevier Science B.V. All rights reserved.

引用

页码：185 / 200

页数：16

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[11]

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[12] Development of an improved product enhanced reverse transcriptase assay [J].