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杨玲焰,王立鹏,荣荣.恒河猴和食蟹猴ABO 血型竞争性等位基因PCR(KASP)检测方法的建立[J].中国比较医学杂志,2019,29(8):31~36.
恒河猴和食蟹猴ABO 血型竞争性等位基因PCR(KASP)检测方法的建立
Establishment of an ABO blood group detection method in Macaca fascicularis and Macaca mulatta using competitive allele specific PCR (KASP)
投稿时间:2019-03-20  
DOI:10.3969/j. issn. 1671 -7856. 2019. 08. 005
中文关键词:  食蟹猴  恒河猴  ABO 血型  竞争性等位基因PCR
英文关键词:Macaca mulatta  Macaca fascicularis  ABO  competitive allele specific PCR
基金项目:
作者单位E-mail
杨玲焰 苏州西山生物技术有限公司,苏州 215123 lingyan.yang @ vrlasia.cn 
王立鹏 苏州西山生物技术有限公司,苏州 215123  
荣荣 西交利物浦大学,苏州 215123 Rong.Rong@ xjtlu.edu.cn 
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中文摘要:
      目的 人的ABO 血型常规免疫学检测方法不适用于实验猴猕猴血型检测,而实验猴血型鉴定在器官移植等研究中必不可少?本研究目的是建立一个快速?可靠的恒河猴与食蟹猴ABO 血型核酸鉴定方法?方法本室建立了一种新的ABO 血型的分子学检测方法,利用竞争性等位基因PCR(KASP)技术,通过识别与血型密切相关功能基因的2 个SNP 位点上的碱基,对恒河猴与食蟹猴血型进行分型,并通过设计2 个SNP 位点外围的普通PCR 引物,对KASP 方法鉴定出的血型样本进行Sanger 测序鉴定?结果 比对了15 份用Taqman PCR 方法已确定血型的食蟹猴全血核酸样本,新建立的KASP 分型结果与原血型结果完全一致,为了保证KASP 方法鉴定结果的准确性,实验人员用新建立的普通PCR 方法通过Sanger 测序验证了KASP 方法与测序结果完全一致?为了对新建的KASP 方法重复性进行验证,选取了已知血型的6 份实验猴全血,每份血样做5 个重复,结果显示该方法有极好的重复性?进一步用新建的KASP 方法检测了51 份临床样本,其中食蟹猴样本38 份,恒河猴样本13 份,同一样本的2 个位点的血型结果完全一致,其中A 型血9 份(17. 6%),B 型血19 份(37. 3%),AB 型血23 份(45. 1%),未检出O 型血?同时从A 型?B 型?AB 型的临床样本中,各型分别选了5 个样本,进行普通PCR 扩增及测序分析,证明2个SNP 位点上的序列与KASP 分型结果一致?结论 新建的KASP 血型鉴定方法准确性高,可用于实验恒河猴与食蟹猴的ABO 血型的快速检测?该方法相较于传统的血清学或测序方法,简单快捷,经济可靠,结果易于判读,同时对样本的要求比传统的血清学方法低,新鲜或存放较久的全血?唾液或核酸等都可以用于检测?
英文摘要:
      Objective Traditional serological method for detecting the ABO blood group in humans are unsuitablefor macaques. Detecting the ABO blood group in macaques is important for transplantation and research. This study aimed toestablish a rapid and reliable nucleic acid-based method for identifying the ABO blood group in Macaca fascicularis andMacaca mulatta. Methods A new molecular method for ABO blood group detection was established, using competitiveallele-specific PCR (KASP) to identify the Macaca fascicularis and Macaca mulatta’ blood group by identifying the basesat 2 single nucleotide polymorphism (SNP) loci that are closely related to the blood-type functional gene. The bloodsamples identified via the KASP method were identified by Sanger sequencing by designing PCR primers around the twoSNP loci. Results Fifteen whole-blood nucleic acid samples from Macaca fascicularis and Macaca mulatta were determinedby TaqMan PCR and compared. The newly established KASP typing result were consistent with the original blood typeresult. To ensure the accuracy of the KASP method identification result, we used the newly established PCR method verifiedby Sanger sequencing, showing that the KASP method was consistent with the sequencing results. To verify the repeatabilityof the new KASP method, six blood samples with known blood groups were selected, and five replicates were taken perblood sample. The results showed that the method had excellent repeatability. In addition, 51 clinical samples were detectedusing the new KASP method, including 38 samples from cynomolgus monkeys and 13 from rhesus monkeys. The blood typeresults for two sites from the same sample were consistent, of which, 9 were type A (17. 6%), 19 were type B (37. 3%),23 were type AB (45. 1%), and none was type O. From the clinical samples with types A, B, and AB, five of each wereselected for PCR amplification and sequencing analysis, which confirmed that the sequences at the two SNP loci wereconsistent with those of the KASP typing. Conclusions The new detection method using KASP is highly accurate and canbe used to rapidly detect the ABO blood types in macaques. Compared with traditional serology and sequencing method, thismethod is simple, rapid, economical and reliable, and the results are easily interpreted. Furthermore, the samplerequirements are lower than those of traditional serological method, and fresh or long-stored whole blood, saliva or nucleic acid can be used.
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