Quality control for DNA contamination in laboratories using PCR-based class II HLA typing methods

Jeffrey M. McCormack, Martha L. Sherman, David H. Maurer

Research output: Contribution to journalArticlepeer-review

5 Scopus citations

Abstract

Quality control (QC) in laboratories performing molecular histocompatibility class II typing often includes a polymerase chain reaction (PCR) approach for monitoring DNA contamination. An oligonucleotide primer set was designed, (RBQBf/RBQBr), which is specific for nonpolymorphic regions of the DR-B, DQ-B, and DP-B consensus sequences with an expected PCR product size of 81 bp. RBQBf/RBQBr detected genomic DNA from reference cell lines LWAGS and BM21 (50 to 100 picograms) as well as DR-B, DP-B, and DQ-B amplicon (1 copy). Additionally, RBQBf/RBQBr detected SSP products from routine DR-B and DQ-B typings. Validation studies employing controlled DNA contamination of laboratory surfaces revealed that increasing amounts of wipe test samples (5% to 20% v/v) were inhibitory to the wipe test PCR, whereas lower amounts (1% to 2%), or alternatively, a diluted wipe test sample, increased the sensitivity of the test and optimized the results. Collectively, this study describes a primer set, RBQBf/RBQBr, which detects both genomic DNA and DR- B, DQ-B, or DP-B amplicon and furthermore illustrates the necessity of routine testing for potential inhibitory factors that may be introduced into the wipe test PCR.

Original languageEnglish (US)
Pages (from-to)82-88
Number of pages7
JournalHuman Immunology
Volume54
Issue number1
DOIs
StatePublished - Apr 15 1997

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