Determination of ABO genotypes by real-time PCR using allele-specific primers

Muro, Tomonori; Fujihara, Junko; Imamura, Seiichiro; Nakamura, Hiroaki; Kimura-Kataoka, Kaori; Toga, Tomoko; Iida, Reiko; Yasuda, Toshihiro; Takeshita, Hitoshi

doi:10.1016/j.legalmed.2011.10.002

Cited by 24 publications

(21 citation statements)

References 25 publications

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“…Yamamoto and his colleagues reported the molecular genetic basis of the ABO group in 1990 [2], permitting analysis of ABO blood groups at the DNA level. Therefore, various PCR-based methods for ABO genotyping have been developed, including PCR-RFLP [3], MS-PCR [4], multiplexed allele-specific PCR [5,6], PCR-SSP [7,8], the universal reporter primer system [9], and real-time PCR using allele-specific primers [10]; these methods have been shown to be useful in forensic investigations. However, most of these methods have shortcomings in terms of the need for post-PCR manipulations, such as enzyme digestion and electrophoresis, and these steps can result in errors and carry-over contamination.…”

Section: Discussionmentioning

confidence: 99%

“…However, most of these methods have shortcomings in terms of the need for post-PCR manipulations, such as enzyme digestion and electrophoresis, and these steps can result in errors and carry-over contamination. PCR-RFLP is the most well-known method for ABO genotyping; however, incomplete restriction enzyme digestion and simultaneous use of restriction enzymes can result in mistyping [10,21]. PCR-SSP requires size separation of PCR fragments by gel electrophoresis and pseudopositive signals from unexpected primer extension reactions often lead to errors in ABO genotyping [8,21,22].…”

Section: Discussionmentioning

confidence: 99%

“…This method is, however, also requires post-PCR manipulations, such as quantification of PCR product and capillary electrophoresis. These post-PCR manipulations may prevent high-throughput analysis and can cause carry-over contamination [10].…”

Section: Discussionmentioning

confidence: 99%

“…Since Yamamoto and colleagues reported the differences in the nucleotide sequences of ABO alleles [1,2], numerous methods for ABO genotyping based on polymerase chain reaction (PCR) have been developed, such as PCR-restriction fragment length polymorphism (PCR-RFLP) analysis [3], mutagenically separated PCR (MS-PCR) [4], multiplexed allele-specific PCR [5,6], PCR with sequence-specific primers (PCR-SSP) [7,8], universal reporter primer systems [9], and real-time PCR using allele-specific primers [10]. However, most of these methods require multiple post-PCR manipulation steps, including electrophoresis and enzyme digestion; for this reason, these methods often have a risk of carry-over contamination [10].…”

Section: Introductionmentioning

confidence: 99%

“…However, most of these methods require multiple post-PCR manipulation steps, including electrophoresis and enzyme digestion; for this reason, these methods often have a risk of carry-over contamination [10].…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

A new method for ABO genotyping using fluorescence melting curve analysis based on peptide nucleic acid probes

Lee¹,

Park²,

An³

et al. 2015

Legal Medicine

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

A new method for ABO genotyping using fluorescence melting curve analysis based on peptide nucleic acid probes

Lee¹,

Park²,

An³

et al. 2015

Legal Medicine

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Rapid ABO genotyping by high‐speed droplet allele‐specific PCR using crude samples

Taira

Matsuda

Takeichi

et al. 2017

Clinical Laboratory Analysis

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Background ABO genotyping has common tools for personal identification of forensic and transplantation field. We developed a new method based on a droplet allele‐specific PCR (droplet‐AS‐PCR) that enabled rapid PCR amplification. We attempted rapid ABO genotyping using crude DNA isolated from dried blood and buccal cells. Methods We designed allele‐specific primers for three SNPs (at nucleotides 261, 526, and 803) in exons 6 and 7 of the ABO gene. We pretreated dried blood and buccal cells with proteinase K, and obtained crude DNAs without DNA purification. Results Droplet‐AS‐PCR allowed specific amplification of the SNPs at the three loci using crude DNA, with results similar to those for DNA extracted from fresh peripheral blood. The sensitivity of the methods was 5%‐10%. The genotyping of extracted DNA and crude DNA were completed within 8 and 9 minutes, respectively. The genotypes determined by the droplet‐AS‐PCR method were always consistent with those obtained by direct sequencing. Conclusion The droplet‐AS‐PCR method enabled rapid and specific amplification of three SNPs of the ABO gene from crude DNA treated with proteinase K. ABO genotyping by the droplet‐AS‐PCR has the potential to be applied to various fields including a forensic medicine and transplantation medical care.

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Rapid and Reliable One-Step ABO Genotyping Using Direct Real-Time Allele-Specific PCR and Melting Curve Analysis Without DNA Preparation

Park

Han

Park

2018

Indian J Hematol Blood Transfus

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ABO genotyping is a molecular diagnostic technique important for transfusion and transplantation in medicine, and human identification in forensic science. Because ABO genotyping are labor intensive and time consuming, the genotyping cannot be firstly used to resolve the serological ABO discrepancy in blood bank. For rapid one-step ABO genotyping, we developed direct, real-time, allele-specific polymerase chain reaction (PCR), and melting curve analysis (DRAM assay) without DNA preparation. In DRAM assay, we used a special PCR buffer for direct PCR, a rapid RBC lysis buffer, white blood cells as template without DNA preparation, allele-specific primers for discriminating three ABO alleles (261G/del, 796C/ A, and 803G/C), and melting curve analysis as a detection method. There was 100% concordance among the results of ABO genotyping by the DRAM assay, serologic typing, PCR-RFLP and PCR-direct sequencing of 96 venous blood samples. We were able to reduce the number of manual steps to three and the hands-on time to 12 min, compared to seven steps and approximately 40 min for conventional ABO genotyping using allele-specific PCR with purified DNA and agarose gel electrophoresis. We have established and validated the DRAM assay for rapid and reliable onestep ABO genotyping in a closed system. The DRAM assay with an appropriate number of allele-specific primers could help in resolving ABO discrepancies and should be valuable in clinical laboratory and blood bank.

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Determination of ABO genotypes by real-time PCR using allele-specific primers

Cited by 24 publications

References 25 publications

A new method for ABO genotyping using fluorescence melting curve analysis based on peptide nucleic acid probes

A new method for ABO genotyping using fluorescence melting curve analysis based on peptide nucleic acid probes

Rapid ABO genotyping by high‐speed droplet allele‐specific PCR using crude samples

Rapid and Reliable One-Step ABO Genotyping Using Direct Real-Time Allele-Specific PCR and Melting Curve Analysis Without DNA Preparation

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