Checklist for optimization and validation of real‐time PCR assays

Journal of Clinical Laboratory Analysis - Tập 23 Số 3 - Trang 145-151 - 2009
Marijke Raymaekers1, Rita Smets1, Brigitte Maes1, Reinoud Cartuyvels1
1Molecular Biology, Clinical Laboratory, Virga Jesse Hospital, Hasselt, Belgium

Tóm tắt

AbstractReal‐time polymerase chain reaction (PCR) is a frequently used technique in molecular diagnostics. To date, practical guidelines for the complete process of optimization and validation of commercial and in‐house developed molecular diagnostic methods are scare. Therefore, we propose a practical guiding principle for the optimization and validation of real‐time PCR assays. Based on literature, existing guidelines, and personal experience, we created a checklist that can be used in different steps of the development and validation process of commercial and in‐house developed real‐time PCR assays. Furthermore, determination of target values and reproducibility of internal quality controls are included, which allows a statistical follow‐up of the performance of the assay. Recently, we used this checklist for the development of various qualitative and quantitative assays for microbiological and hematological applications, for which accreditation according to ISO 15189:2007 was obtained. In our experience, the use of the proposed guidelines leads to a more efficient and standardized optimization and validation. Ultimately, this results in reliable and robust molecular diagnostics. The proposed checklist is independent of environment, equipment, and specific applications and can be used in other laboratories. A worldwide consensus on this kind of checklist should be aimed at. J. Clin. Lab. Anal. 23:145–151, 2009. © 2009 Wiley‐Liss, Inc.

Từ khóa


Tài liệu tham khảo

10.1126/science.2999980

10.1016/0076-6879(87)55023-6

10.1038/nbt0492-413

10.1038/nbt0993-1026

10.1073/pnas.88.16.7276

10.1093/nar/21.16.3761

10.1101/gr.4.6.357

10.1016/j.cccn.2005.07.009

NCCLS, 2003, Quantitative molecular methods for infectious diseases: Approved guidelines, 1

10.1016/j.jcv.2005.12.006

10.1016/j.clinmicnews.2007.05.002

10.1016/j.jcv.2007.07.009

Organization Economic Cooperation and Development (OECD) OECD guidelines for quality assurance in molecular genetic testing;http://www.oecd.org/dataoecd/43/38839788.pdf.

AmosJ FeldmanG WayneW.Technical standards and guidelines for CFTR mutation testing.2006.

College of American Pathologists. Laboratory accreditation program checklist Laboratory accreditation program accreditation checklists.2008.

10.1016/S0301-472X(02)00806-8

10.1039/a708608c

10.1016/S1074-5521(00)00058-2

10.1093/nar/28.2.655

10.1038/nbt0198-49

10.1038/nbt0396-303

Caplin BE, 1999, Lightcycler hybridization probes—the most direct way to monitor PCR amplification and mutation detection, Biochemistry, 1, 5

Kusser W, 2006, Use of self‐quenched, fluorogenic LUX primers for gene expression profiling, Methods Mol Biol, 335, 115

10.1006/abio.2000.4534

10.1093/nar/30.2.e5

10.1016/j.mam.2005.12.007

10.1586/14737159.5.2.209

Aslanzadeh J, 2004, Preventing PCR amplification carryover contamination in a clinical laboratory, Ann Clin Lab Sci, 34, 389

Bustin SA, 2004, Pitfalls of quantitative real‐time reverse‐transcription polymerase chain reaction, J Biomol Tech, 15, 155

10.1111/j.1198-743X.2004.00722.x

10.1586/14737159.4.2.219

Muller MC, 2007, Harmonization of BCR‐ABL mRNA quantification using a uniform multifunctional control plasmid in 37 international laboratories, Leukemia

10.1038/sj.leu.2403590

10.1007/978-1-4612-0055-0_19

AppliedBiosystems. Primer Express Software;http://primer‐express.software.informer.com

Rozen S, 2000, Bioinformatics Methods and Protocols: Methods in Molecular Biology, 365

10.1007/978-1-59745-528-2_2

10.1093/nar/16.20.9869

10.1007/978-1-349-20235-5_1

Brooks EM, 1995, Secondary structure in the 3' UTR of EGF and the choice of reverse transcriptases affect the detection of message diversity by RT‐PCR, Biotechniques, 19, 806

Suggs SV, 1981, ICN‐UCLA Symposium on Developmental Biology, 683

10.1073/pnas.48.8.1390

10.1073/pnas.83.11.3746

10.1073/pnas.83.24.9373

10.1002/(SICI)1098-2825(1996)10:5<277::AID-JCLA8>3.0.CO;2-5

10.1093/nar/17.21.8543

10.2144/97221bi01

10.1002/(SICI)1098-2825(1996)10:5<285::AID-JCLA9>3.0.CO;2-7

10.1006/abio.2000.4547

10.1093/nar/26.5.1167

10.3109/10409239109114071

Newton CR, 1994, PCR

Compliance C.s.C.L.R.A.g.t.C. Washing G‐2 Reports.1997. Washington.

10.1128/CMR.19.1.165-256.2006

10.1385/MB:26:2:133

10.1111/j.1600-0463.2004.apm11211-1207.x

10.1677/jme.0.0250169

10.1006/meth.2001.1262

10.1093/nar/29.9.e45

10.1093/nar/30.9.e36

10.1016/S0966-3274(03)00010-8

10.1023/B:BILE.0000019559.84305.47

10.1038/sj.gene.6364190

NCBI http://www.ncbi.nlm.gov/blast/Blast.cgi.

10.1093/nar/gkl1031

10.1073/pnas.87.6.2264

10.1016/S0022-2836(05)80360-2

10.1007/978-3-642-59524-0_3

10.2144/05391RV01

10.1186/1471-2105-7-85

10.1006/abio.2001.5530

10.1023/A:1021319421153

10.1016/j.mcp.2003.09.001

10.1093/nar/gnh177

10.1093/nar/gng122

Cikos S, 2007, Relative quantification of mRNA: Comparison of methods currently used for real‐time PCR data analysis, Mol Biol, 8, 113

10.1111/j.1556-4029.2006.00182.x

Snedecor G, 1980, Statistical Methods

CLSI CLSI document MM3‐A2.

CLSI CLSI document CS2.

Westgard Tietz textbook of clinical chemistry.1999.

CLSI CLSI document M14A.

CLSI CLSI document GP29A.

10.1038/sj.leu.2404388

10.1182/blood-2006-01-0092

Thông tin
Thông tin xuất bản

Journal of Clinical Laboratory Analysis

Tập 23 Số 3

145-151

Thông tin tác giả