演題詳細

ポスター / Poster

【E】ポスター 43 (Poster 43) :GVHD/Chimerism

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日程
2013年10月11日(金)
時間
16:50 - 17:50
会場
ポスター会場 / Poster (ロイトン札幌 3F エメラルドABCD)
座長・司会
岡本 康裕 (Yasuhiro Okamoto):1
1:Department of Pediatrics, Kagoshima University Hospital, Japan
 
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Fast STR-PCR protocol enabling rapid and high quality chimerism analysis after allo-HSCT

演題番号 : PS-1-330

Hongxing Liu (刘 红星):1、Qian Niu:1、Fang Wang:1、Wen Teng:1、Yan Wang:1、Jiangyan Fan:1、Chunrong Tong:1

1:Lu Daopei Hematology & Oncology Center, Yanda International Hospital, China

 

Background: Chimerism analysis based on STR-PCR is the standard method for chimerism analysis after allogeneic hematopoietic stem cell transplant (allo-HSCT). The cycling time needs about 3.5 hours, and incomplete adenylation of PCR products often become a problem. Here we aimed to develop a fast PCR protocol enables rapid and high quality chimerism analysis. Methods: Peripheral blood and fingernail samples were collected from healthy donors and patients. Simulated mixed chimerism samples were prepared by fold dilution. The AB Identifiler Kit with 15 STR loci was used. AmpliTaq Gold polymerase and standard PCR cycling program were used as contrast. Newly bio-engineer modified fast polymerase MyTaq, Phusion and Q5 were tested with rapid cycling programs. The PCR products were capillary electrophoresed and chimerism was analyzed using AB3500XL and GeneMapper IDX software. Results: All the four kinds of polymerase can be successfully used for STR-PCR and genotyping. The STR-PCR cycling time for MyTaq, Phusion and Q5 were about 30 minutes, considerably less than 3.5 hours when using AmpliTaq Gold. For the 3 kinds of fast polymerases, the Q5 polymerase showed the best balanced amplification efficiency among the 15 STR loci. The PCR product of Phusion and Q5 don’t have non-template addition “A” tail due to lack of adenylate activity, yet the allelic ladders without “A” tail must be specially made. The MyTaq has extremely efficient of adenylate activity and showed complete adenylation even using crude DNA samples extracted from fingernail, thus actually fully resolved the problem of incomplete adenylation. Conclusion: The STR-PCR cycling time got a drastic reduction and the problem of incomplete adenylation was resolved by using the novel MyTaq and rapid PCR program, thus enabling rapid and high quality chimerism analysis after allo-HSCT.

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