HLA‐B*5701 typing: evaluation of an allele‐specific polymerase chain reaction melting assay

Abstract

Inheritance of HLA-B*5701 is a strong predictor of a hypersensitivity reaction to the anti-HIV drug abacavir. The identification of susceptible individuals prior to the institution of abacavir therapy is therefore of clinical importance and has generated demand for a simple and rapid diagnostic test for carriage of HLA-B*5701. In this study, we describe the development of such a method based on allele-specific polymerase chain reaction (AS-PCR) and melting curve analysis. Ninety-six patient samples including 36 HLA-B*5701-positive samples and 60 HLA-B*5701-negative samples were analysed. Compared with sequence-based typing, this method had 100% sensitivity and specificity for the HLA-B*5701 allele. In conclusion, the AS-PCR/melting curve approach minimises post-polymerase chain reaction handling processing and provides an attractive alternative to currently described AS-PCR methods.

Determination of the High Prevalence of Dual/Mixed- or X4-Tropism Among HIV Type 1 CRF01_AE in Hong Kong by Genotyping and Phenotyping Methods

“…For instance, deletion of residues 9 to 12 affects tropism and the extracellular loops of the CXCR4 coreceptor interact more easily with the longer V3 loop. 34,35 Yet none of these effects was observed or had an association with the discordant genotypic coreceptor predictions as suggested. 36 …”

Novel Sensitive, Specific and Rapid Pharmacogenomic test for the Prediction of Abacavir Hypersensitivity Reaction: HLA-B*57:01 Detection by Real-Time PCR

“…In a subsequent study, the same research group described a faster test carried out in Q-PCR [19]. For this approach, they used only three of the four HLA-B*57:01 SSPs previously characterized [12], thus missing the opportunity to discriminate the HLA-B*57:06 allele [19]. Briefly, the authors used together with the HLA-B*57:01-specific primers, a set of primers specific for the housekeeping gene, the HGH gene, that yielded a 439-bp product.…”

“…The semiquantitative PCR technique has been progressively replaced by Q-PCR, a more rapid and sensitive method for the direct monitoring of DNA amplification during PCR cycling. In a subsequent study, the same research group described a faster test carried out in Q-PCR [19]. For this approach, they used only three of the four HLA-B*57:01 SSPs previously characterized [12], thus missing the opportunity to discriminate the HLA-B*57:06 allele [19].…”

“…The authors set up a multiplex Q-PCR to favor amplification of the HLA-B*57:01-specific product when the allele is present instead of the amplification of the HGH-specific amplicon. The assessment of the HLA-B*57:01 status was carried out by analyzing differences in the melting curves of the two different amplified products [19]. However, our preliminary data demonstrated a significantly lower Q-PCR efficiency and sensitivity when both primer sets were used directly on crude DNA preparation (data not shown); therefore, HLA-B-specific amplification using already validated primers was introduced in our strategy [16].…”

HLA and Pharmacogenetics of Drug Hypersensitivity

“…When considering the laboratory designation of HLA typing, several alternative approaches can successfully be adopted. In the case of HLA-B*5701 for example, simple PCR methods including simple sequence-specific (SSP)-PCR, sequencebased typing or real-time PCR melt curve analysis are all applicable [37,[130][131][132][133]. Sequencebased typing or allele-specific PCR are the most definitive means of identifying the presence of HLA-B*5701; however, due to greater ease of use some clinical laboratories choose to perform SNP testing over allele-specific PCR.…”