Comparison of a Simple-Probe Real-Time PCR and Multiplex PCR Techniques for HPA-1 to HPA-6 and HPA-15 Genotyping

Abstract

Background We aimed to compare HPA‐1 to HPA‐6 and HPA‐15 genotyping results obtained by a simple‐probe real‐time polymerase chain reaction (PCR) technique with the multiplex PCR technique. Methods Five hundred DNA samples from the Thai National Stem Cell Donor Registry (TSCDR) of the National Blood Centre, Thai Red Cross Society were included. Human platelet antigen (HPA) genotyping was performed by simple‐probe real‐time PCR and multiplex PCR techniques. Results HPA‐1, HPA‐2, HPA‐3, and HPA‐4 genotyping results obtained by both techniques were in agreement. The misinterpretation of HPA‐5, HPA‐6, and HPA‐15 genotypes was found in eight samples by simple‐probe real‐time PCR and HPA genotypes were confirmed by DNA sequencing. Two samples of HPA‐5 were misinterpreted as HPA‐5a5a instead of HPA‐5a5b due to an NM_002203.3:c.1594A>C mutation (rs199808499) near the HPA‐5 polymorphism (5′ side). Five samples of HPA‐6a6b were misinterpreted as HPA‐6b6b because of an NM_000212.2:c.1545G>A mutation (rs4634) adjacent to the HPA‐6 polymorphism (3′ side). Interestingly, one sample of HPA‐15a15b was misinterpreted as HPA‐15b15b due to an NM_133493.1:c.2118C>A mutation near the HPA‐15 polymorphism (3′ side). Conclusions HPA genotyping results by two PCR techniques were compared. Incorrect assignments were found due to genetic variations near each HPA single nucleotide polymorphism. Therefore, to avoid false assignation, the use of two genotyping techniques is recommended.

Simultaneous genotyping of human platelet alloantigen‐1 to 28bw systems by multiplex polymerase chain reaction sequence‐based typing

“…The validity of the Hardy–Weinberg (HW) equilibrium was tested by calculating expected numbers of subjects for each genotype as described previously . Agreement of the observed and expected genotype, based on the HW equilibrium, was determined using the chi‐square test.…”

“…prevention and treatment of patients with immune platelet disorders and for the provision of compatible blood products from HPA-typed donor panel populations. Currently, DNA-based methods have been widely used for HPA genotyping [8][9][10][11][12][13], such as PCR sequence-specific primers (PCR-SSP) [8], PCR sequence-based typing (PCR-SBT) [9], BeadChip Microarray Technology [10], real-time PCR [11], matrix-assisted laser desorption/ionization time-of-flight mass spectrometry [12,13]. We have also reported a PCR-SBT assay combining with high-throughput DNA sequencer for identifying the genotypes of HPA-1 to 17w systems [14].…”

A Novel SimpleProbe PCR Assay for Detection of Mutations in the 23S rRNA Gene Associated with Macrolide Resistance in Mycoplasma genitalium in Clinical Samples

“…Our aim was to develop a simple and sensitive method for diagnostic use to differentiate between the macrolide-susceptible wild type and mutant types of M. genitalium without the need for nucleotide sequence determination. The method has been used for the detection of other microbes ( 20 , 21 ) but, to our knowledge, has never been applied to M. genitalium mutation detection. The study also aimed to present the prevalence of macrolide-resistant M. genitalium isolates in a Norwegian population.…”

Approach to Neonatal Alloimmune Thrombocytopenia: The Perspective from a Transfusion Medicine Service

“…73 (a) Sequence-specific primer-polymerase chain reaction: This is an allele-specific PCR that uses two reactions, using two sets of primers; one is specific for each allele and the second control primer used to monitor the efficiency of PCR. 74,75 When there is 3'-terminal nucleotide mismatch between the allele-specific primer and the target DNA, there may be some loss of efficiency of Taq polymerase in DNA amplification and this forms the basis of SSP-PCR. The HPA profile is identified by the presence or absence of DNA bands that appear after gel electrophoresis of the products obtained by PCR.…”