Development and evaluation of a real-time quantitative PCR for the detection of human cytomegalovirus

被引：50

作者：

Kearns, AM

Guiver, M

James, V

King, J

机构：

[1] Newcastle Gen Hosp, Publ Hlth Lab, Newcastle Upon Tyne NE4 6BE, Tyne & Wear, England

[2] Withington Hosp, Publ Hlth Lab, Manchester M20 2LR, Lancs, England

[3] Cent Publ Hlth Lab, London NW9 5HT, England

[4] Quality Assurance Lab, London NW9 5HT, England

[5] Publ Hlth Lab, Exeter EX2 5AD, Devon, England

来源：

JOURNAL OF VIROLOGICAL METHODS | 2001年 / 95卷 / 1-2期

关键词：

cytomegalovirus; viral load; real-time PCR; quantitative PCR; transplant patients;

D O I：

10.1016/S0166-0934(01)00307-X

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

A novel real-time quantitative PCR (QPCR) assay is described for monitoring CMV DNA load in clinical specimens using the LightCycler (TM). The assay is rapid (< 40 min), easy to carry out, robust, reliable and is capable of detecting from 10 to over 2 x 10(5) CMV DNA copies with a wide linear range. Amplification and detection occur simultaneously. avoiding the need for post-PCR analysis and thereby minimising the risk of carryover contamination. The assay proved to be accurate, specific and reproducible when evaluated in three different laboratories. In addition. LightCycler (TM) results were comparable with those of TaqMan (TM). an independent real-time QPCR assay. (C) 2001 Elsevier Science B.V. All rights reserved.

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页码：121 / 131

页数：11

相关论文

共 23 条

[1] Use of molecular assays in diagnosis and monitoring of cytomegalovirus disease following renal transplantation [J].

Aitken, C ;

Barrett-Muir, W ;

Millar, C ;

Templeton, K ;

Thomas, J ;

Sheridan, F ;

Jeffries, D ;

Yaqoob, M ;

Breuer, J .

JOURNAL OF CLINICAL MICROBIOLOGY, 1999, 37 (09) :2804-2807

[2]

ANAKA N, 2000, J MED VIROL, V60, P455

[3]

[Anonymous], 1990, J BUSINESS TECHNICAL

[4] Integrated amplification and detection of the C677T point mutation in the methylenetetrahydrofolate reductase gene by fluorescence resonance energy transfer and probe melting curves [J].

Bernard, PS ;

Lay, MJ ;

Wittwer, CT .

ANALYTICAL BIOCHEMISTRY, 1998, 255 (01) :101-107

[5] A highly sensitive assay for detection and quantitation of human cytomegalovirus DNA in serum and plasma by PCR and electrochemiluminescence [J].

Boom, R ;

Sol, C ;

Gerrits, Y ;

De Boer, M ;

Wertheim-van Dillen, P .

JOURNAL OF CLINICAL MICROBIOLOGY, 1999, 37 (05) :1489-1497

[6] Use of real-time PCR and fluorimetry to detect lamivudine resistance-associated mutations in hepatitis B virus [J].

Cane, PA ;

Cook, P ;

Ratcliffe, D ;

Mutimer, D ;

Pillay, D .

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, 1999, 43 (07) :1600-1608

[7] USE OF THE POLYMERASE CHAIN-REACTION TO ANALYZE SEQUENCE VARIATION WITHIN A MAJOR NEUTRALIZING EPITOPE OF GLYCOPROTEIN-B (GP58) IN CLINICAL ISOLATES OF HUMAN CYTOMEGALOVIRUS [J].

DARLINGTON, J ;

SUPER, M ;

PATEL, K ;

GRUNDY, JE ;

GRIFFITHS, PD ;

EMERY, VC .

JOURNAL OF GENERAL VIROLOGY, 1991, 72 :1985-1989

[8] Application of viral-load kinetics to identify patients who develop cytomegalovirus disease after transplantation [J].

Emery, VC ;

Sabin, CA ;

Cope, AV ;

Gor, D ;

Hassan-Walker, AF ;

Griffiths, PD .

LANCET, 2000, 355 (9220) :2032-2036

[9] LONGITUDINAL ANALYSIS OF CYTOMEGALOVIRUS LOAD IN RENAL-TRANSPLANT RECIPIENTS USING A QUANTITATIVE POLYMERASE CHAIN-REACTION - CORRELATION WITH DISEASE [J].

FOX, JC ;

KIDD, IM ;

GRIFFITHS, PD ;

SWENY, P ;

EMERY, VC .

JOURNAL OF GENERAL VIROLOGY, 1995, 76 :309-319

[10] CYTOMEGALO-VIRUS AS A RISK FACTOR IN RENAL-TRANSPLANTATION [J].

FRYD, DS ;

PETERSON, PK ;

FERGUSON, RM ;

SIMMONS, RL ;

BALFOUR, HH ;

NAJARIAN, JS .

TRANSPLANTATION, 1980, 30 (06) :436-439