Real-time PCR genotyping of human platelet alloantigens HPA-1, HPA-2, HPA-3 and HPA-5 is superior to the standard PCR-SSP method

Genotyping of the human platelet alloantigens (HPA) is useful for the diagnosis and therapy of the patients with alloimmune thrombocytopenic syndromes, such as post-transfusion refractoriness to platelets, post-transfusion thrombocytopenic purpura and foetomaternal alloimmune thrombocytopenia. We have developed, optimized and validated a new method for simultaneous genotyping of HPAs - HPA-1, HPA-2, HPA-3 and HPA-5 - by using the real-time polymerase chain reaction (PCR) based on TaqMan technology. Its performances were compared to those of the standard PCR-sequence-specific primers (SSP) method by testing 120 DNA samples. Several discrepancies between the two methods have been observed, especially in the HPA-3 genotyping. Evidently, the PCR-SSP method produced several false positive results due to its technical drawbacks. Based on our comparison, we believe that the new real-time TaqMan PCR assay for the HPA-1, HPA-2, HPA-3 and HPA-5 genotyping is faster, more reliable and reproducible, compared to the standard PCR-SSP.     

使用Taqman PCR技术建立人类血小板抗原-1～5、15分型体系

目的了解上海地区单采血小板献血人群HPA-1～5、15多态性分布,分析评估新的分型技术。方法利用TaqMan PCR技术对500份上海地区单采血小板供者标本进行HPA-1～5、15抗原系统等位基因分型,并随机抽取100份标本使用PCR-SSP技术进行比对。结果 HPA各等位基因频率分别为HPA-1a:0.999,HPA-1b:0.001,HPA-2a:0.953,HPA-2b:0.047,HPA-3a:0.582,HPA-3b:0.418,HPA-4a:0.999,HPA-4b:0.001,HPA-5a:0.988,HPA-5b:0.012,HPA-15a:0.524,HPA-15b:0.476;有1份标本HPA-5等位基因与SSP检测结果产生差异。结论上海地区HPA各等位基因频率与国内各地区人群分布无明显差异,与中国汉族人群HPA分布情况基本吻合,实验数据经验证符合Hardy-Weinberg平衡定律,实验结果准确可靠;HPA-5差异经测序验证判断可能由PCR-SSP非特异扩增所致;TaqMan技术在HPA抗原系统分型应用中特异性高,反应时间短,具有良好的应用前景,是现有技术方法的一种重要补充。     

一种多重PCR方法快速鉴定7种常见肠道病原菌

目的 建立一种能够快速检测并分型5种致泻性大肠埃希菌、志贺菌及沙门菌的多重聚合酶链反应(multiplex polymerase chain reaction,M-PCR) 方法。 方法 设计针对7种常见肠道致病菌的12对引物,通过多重PCR方法扩增后电泳观察相应条带,确定病原菌。临床标本直接划线接种于肠道选择性平皿,挑取可疑菌落直接提取核酸进行多重PCR检测。 结果 所有标准菌株均能扩增到相应目的片段,322份腹泻病标本中使用该方法共检出志贺菌24株(福氏志贺菌7株、宋内志贺菌17株)、 沙门菌5株、EPEC共12株(均为aEPEC)、ETEC共6株(elt阳性3株;est阳性3株)、EIEC共3株、VTEC共1株(stx1和stx2A均阳性)、EAEC共3株。 结论 该方法快速特异,能同时检测7种常见肠道致病菌,可用于腹泻病常见病原的快速检验。     

青岛地区汉族人群HPA-1—5,15多态性分布研究

目的研究青岛地区汉族人群人类血小板抗原(HPA)1-5,15抗原分布多态性。方法采用PCR-SSP方法对青岛地区918名无血缘关系固定血小板无偿捐献者进行HPA1-5及HPA-15系统的基因分型.结果各被检系统等位基因频率分别是1a=0.9940,1b=0.0060,2a=0.9319,2b=0.0681,3a=0.5822,3b=0.4178,4a=0.9897,4b=0.0104,5a=0.9804,5b=0.0196,15a=0.4913,15b=0.5087;HPA基因频率分布与国内资料比较,HPA-1与北方人群(河南),HPA-2与南方人群(四川)差异有统计学意义;与台湾人群HPA-2,-4,与日本人群HPA-2,-3,-5,与美国黑人HPA-1,-2,-5,与白人HPA-1,-4,-5,-15分别有统计学显著性差异。结论青岛地区汉族人群HPA分布具有本地人群特点。本组HPA数据分布符合Hardy-Weinberg平衡定律,可以作为北方汉族人群HPA基因分布频率数据库和青岛本地化血小板供者HPA资料库。     

人类血小板抗原HPA-15系统PCR-SSP基因分型技术的建立

本研究目的是采用PCR-SSP技术建立人类血小板抗原HPA-15系统的基因分型方法,并应用于血小板供者库的HPA基因定型。采用第11届国际输血协会（ISBT）血小板血清学与基因分型协作组推荐的序列特异性引物,调节引物浓度、Mg2＋离子浓度和探索最佳PCR扩增条件,建立HPA-15系统基因分型技术。该分型技术的准确性和可靠性,采用第11届ISBT血小板协作组提供的质控样本进行验证,同时采用本研究合成的引物及商品化试剂盒,对50名随机的汉族血小板捐献者进行HPA-15系统基因分型,作为平行对照。应用本研究的方法,对第11届ISBT送检的10份考核样本进行基因分型。结果表明：基因分型结果与ISBT公布的结果完全一致。50名随机的血小板志愿捐献者,经本研究的方法及美国G＆T公司的试剂检测,基因分型的结果相符合;观察到的基因频率：HPA-15a和-15b分别为0.5100和0.4900。结论：本研究建立的HPA基因分型技术具有简便、快速、准确的特点,适合于常规HPA基因分型,具有广泛的应用前景。     

浙江汉族人群血小板抗原1-16多态性调查

目的了解浙江汉族人群血小板抗原1-16多态性分布。方法采用PCR-SSP方法对120份浙江无血缘关系汉族样本作HPA-1—16等位基因分型。结果HPA-1b、-2b、-3b、-6bw、-15b的基因频率分别为0.0125、0.0542、0.3833、0.0125、0.4833,未检出HPA-4b、-5b、-7bw、-8bw、-9bw、-10bw、-11bw、-12bw、-13bw、-14bw、-16bw等位基因。浙江汉族人群HPA-3、15抗原系统血小板随机输注错配概率分别为0.36、0.37。结论浙江汉族人群HPA多态性分布与中国其他地区汉族人群比较没有差异,HPA-3和15系统多态性较丰富,因此在随机血小板输注或胎母之间比较容易发生同种免疫反应。     

多重PCR同时检测病原菌及其耐药基因

目的建立一种多重PCR方法,在同一扩增体系内同时检测病原菌及其β-内酰胺类耐药基因,探讨采用多重PCR同时鉴定病原菌及其耐药基因的可行性。方法根据细菌基因序列和对β-内酰胺类抗生素耐药特点,设计一对细菌鉴定通用引物和三对β-内酰胺类耐药基因引物,并在同一扩增体系内行PCR扩增。结果 MRSA和大肠埃希菌及肺炎克雷伯菌的产酶标准菌株的bla mecA、blaTEM、blaSHV和16S-23S rRNA基因间隔区（ISR）多重扩增均为阳性,对本实验室内保存的50株多重耐药的葡萄球菌（包括40株表型筛查为MRS菌株及10株非MRS菌株）及30株ESBLs阳性的大肠埃希菌及30株肺炎克雷伯菌的多重PCR结果显示：MRS阳性菌株均同时扩增出了葡萄球菌特有的多态性及blamecA指纹图谱,而多重耐药的非MRS菌株也都扩增出了blamecA,ESBLs表型阳性的大肠埃希菌多以blaTEM为主,而肺炎克雷伯菌多以blaSHV为主。结论多重PCR与传统的培养及药敏试验相比敏感、特异、迅速,对于解决难培养或不能培养的微生物的鉴定和药敏试验,是一种很有前景的方法。     

Genotyping of 38 insertion/deletion polymorphisms for human identification using universal fluorescent PCR

Short insertion/deletion (Indel) polymorphisms of approximately 2–6 bp are useful as biallelic markers for forensic analysis, and the application of Indel genotyping as a supplementary tool would improve human identification accuracy. We examined the allele frequencies of 37 autosomal Indels in the Japanese population and developed a novel dual-color genotyping method for human identification on the basis of universal fluorescent PCR, including the sex-typing amelogenin locus. Target genomic fragment sizes for 38 Indels were 49–143 bp. We analyzed these Indels in 100 Japanese individuals using the M13(-47) sequence as a universal primer. For dual-color genotyping, we designed a novel universal primer with high amplification efficiency and specificity. Using FAM-labeled M13(-47) and HEX-labeled modified M13(-47) primers, fluorescent signals at all loci were clearly distinguished in two independent multiplex PCRs. Average minor allele frequency was 0.39, and accumulated matching probability was 2.12 × 10⁻¹⁵. Complete profiles were successfully amplified with as little as 0.25 ng of DNA. This method provides robust, sensitive, and cost-effective genotyping for human identification.     

多重实时荧光PCR快速检测沙门菌和单增李斯特菌

目的 建立可同时检测沙门菌、单增李斯特菌的双重实时荧光PCR快速检测方法,并应用于食品样品的检测.方法 根据GenBank上下载的沙门菌invA基因、单增李斯特菌hlyA基因的保守区序列分别设计特异性引物和TaqMan探针,建立并优化双重实时荧光PCR反应体系.对该方法的特异性、敏感性和稳定性进行评估,并应用于食品标本...     

大理地区志贺菌的血清型分布及多重PCR检测技术的应用

目的 探讨大理地区志贺菌的血清型分布以及多重PCR技术在志贺菌检测中的应用.方法 用常规分离培养、生化鉴定和血清学分型法对腹泻患者粪标本进行检测,分离到的标本再用多重PCR法对志贺菌的毒力基因shetA、shetB、ial和ipaH进行扩增检测.结果 通过常规培养和生化反应鉴定,得到68株志贺菌.血清学鉴定显示福氏志贺菌41株,宋内志贺菌25株,鲍氏志贺菌2株.通过多重PCR扩增后68株志贺菌均在423 bp(ipaH)处出现了条带,检出率为100%,其余在147 bp(shetB)、309 bp(shetA)处以及320 bp(ial)处均出现了至少一个条带,与常规鉴定法的符合率达100%.5份直接用阳性粪便标本提取DNA为模板进行多重PCR扩增后,也得到了同样的结果.结论 大理地区细菌性痢疾患者感染以福氏志贺菌为主,多重PCR技术可用于志贺菌的快速检测和流行病学调查.     

Analysis of RHD genes in Taiwanese RhD-negative donors by the multiplex PCR method

The determination of the RhD phenotype is important in transfusion medicine. However, due to the complexity of D antigen expression, the routine serological method cannot differentiate all RhD variants. In addition, the induction of the anti-D antibody is still the major cause of severe hemolytic disease of the newborn (HDN). Therefore, it is important to understand RHD gene profiles. To analyze the RHD gene profiles of Taiwanese RhD-negative donors, the multiplex PCR method was applied to amplify RHD specific exons 3, 4, 5, 7, and 9. Based on the PCR results, the 156 RhD-negative donors were divided into 12 groups according to the different expression patterns of the RHD gene. These 12 groups were further divided into three categories: type I=Rh D(el) (21.8%); type II = partial D, containing some exons (9.0%); and type III = true RhD-negative (69.2%). The results indicated that 21.8% of RhD-negative donors in Taiwan were RhD(el), and 9% carried a part of the RHD gene. Six defined RhD variants were found in this study: four R(O) (Har), one D(Va), and two D(IVb). However, no true RhD-negative or RhD(el) donor with the CcdEe phenotype was found in this analysis.     

海南黎族人血小板1—17抗原基因与随机输血不配合率研究

目的调查海南岛黎族人群血小板抗原(HPA)-1—17等位基因多态性及其特点,评估其在随机输血中血小板输注无效的风险。方法采用PCR-SSP方法对海南岛180名黎族人群做HPA-1—17基因分型检测>计算各系统对偶抗原不配合率。结果在海南黎族人群的HPA-1—17系统中,呈多态性分布的等位基因及其频率为HPA-2a(0.9972)、2b(0.0028),HPA-3a(0.4889)、3b(0.5111),HPA-5a(0.9667)、5b(0.0333),HPA-6a(0.9972)、6b(0.0028),HPA-15a(0.4250)、15b(0.5750);其余HPA-1、4、7—14、16、17等位基因均呈单线性分布。在HPA-3、15等位基因中出现bb纯合子基因型,频率分别为0.2834和0.3667;其余系统均未见bb纯合子基因型。随机输血中,海南岛黎族人群HPA不配合的发生率依次为:HPA-3(37.49%)、-15(36.93%)和-5(6.23%)。结论揭示了海南岛黎族人HPA-1—17基因型和等位基因频率的分布及特点;立足人群的随机血小板输注,只需检测供、受者HPA-2,-3、-5和-15基因相合,就可基本达到血小板匹配性输注。     

Detection of MNS hybrid molecules in the Thai population using PCR-SSP technique

We developed a polymerase chain reaction-sequence-specific primer (PCR-SSP) technique to screen for hybrid molecules in the MNS blood group in the Thai population using two sets of newly designed primers specific for four GYP(B-A-B) hybrids, GP.Mur, GP.Hop, GP.Bun and GP.HF, and two GYP(A-B-A) hybrids, GP.Vw and GP.Hut. One thousand and forty-one blood samples were tested with human anti-Mi(a) by conventional tube technique, and 598 samples of these were tested by the PCR-SSP technique. Ninety-four samples (9.03%) were strongly positive with human antisera by conventional tube technique. For PCR-SSP test results, the GP.Hut, GP.Mur, GP.Hop, GP.Bun and GP.HF genotypes were amplified with the first set of primers, whereas GP.Vw genotype was amplified with a second set of primers. The GYP(A-B) hybrids (GP.Hil and GP.JL), GYP(A-B-A) hybrids (GP.Nob, GP.Joh and GP.Dane), GYPA, GYPB and GYPE were not amplified by either set of primers. Results of testing 94 Mi(a+) and 504 Mi(a-) by conventional tube technique and PCR-SSP were concordant. This study shows that analysis by PCR-SSP is simple and convenient; therefore, it can be used as an alternative to conventional tube technique for mass screening for MNS hybrids, especially when specific antisera are not available.     

多重PCR在幽门螺杆菌检测及分型中的应用

应用多重ＰＣＲ同时扩增１６ＳｒＲＮＡ和ｃａｇＡ基因来检测幽门螺杆菌感染及其分型。ＰＣＲ产物经ＤＮＡ序列测定证实为转异性扩增，敏感度为１０＾２ＣＦＵ／ｍｌ。４８株Ｈｐ菌株中，Ｉ型菌株（ｃａｇＡ＋）２６株（５４．２％）；５５０份胃粘液标本，Ｈｐ阳性（１６ＳｒＲＮＡ基因＋）２１６份（４７．５％），其中Ｉ型Ｈｐ１３９份（５３．３％）。     

Frequency distribution of human platelet antigens in the Indian population

This study was undertaken with an aim of establishing the frequency distribution of various human platelet antigens (HPA) in Indian populations by means of DNA-based technology. A total of 1164 people belonging to various population groups were studied for the frequency distribution of HPA. DNA extraction was performed from peripheral venous blood samples. Polymerase chain reaction allele-specific amplification technique was used for HPA genotyping. The HPA bands were visualized by using ethidium bromide-stained agarose gel, after electrophoresis. The homozygosity of the HPA-1b/1b genotype was found to be significantly higher (P < 0.05) in the Parsi population group and Vatalia Prajapati population group, compared to Maharashtrians. Frequency distribution of HPA-1b in our populations was found to be slightly lower than that reported in some western populations. This study has established a DNA technique to diagnose cases of NAITP definitively and to treat these cases during the neonatal period, and also gives the frequency distribution of HPA in some of the Indian population.     

多重实时荧光定量PCR方法在高危型人乳头瘤病毒E6/E7 mRNA检测中的应用

摘要：目的?建立一种基于多重实时荧光定量PCR(MRT-PCR)技术的可同时检测14种高危亚型人乳头瘤病毒(HPV16、18、31、33、35、39、52、45、51、56、58、59、66、68)癌基因E6/E7 mRNA的方法。方法?对14种高危型HPV各自的E6/E7基因序列区域设计特异性引物和探针，配以优化的反应体系，形成HPV E6/E7 mRNA检测体系，评价方法检出限、特异性。收集223例临床样本，分别用自建的一步法MRT-PCR法与转录介导等温核酸扩增(TMA)技术(Aptima?HPV试剂盒)检测HPV E6/E7 mRNA，同时评估HPV E6/E7 mRNA检测结果与薄层液基细胞学检测、宫颈组织病理学检测结果的一致性。结果?建立的检测方法对常见的低危型HPV无交叉检出，且对14种高危型HPV的检测限可达10～100 copies/μL。该方法检测223例临床标本的结果与Aptima??HPV检测结果相比，一致性较好(Kappa=0.910)。以组织病理结果为CINⅡ及以上的作为阳性，MRT-PCR法临床检测敏感性为84.0%，特异性为94.4%，阳性预测值为65.6%，阴性预测值为97.9%。结论?建立的MRT-PCR方法具有特异性好、灵敏度高和稳定性好等优点，为HPV临床快速检测以及宫颈病变筛查提供了一个有效的技术平台。     

人类血小板抗原1—16基因与血小板输注无效风险研究

目的探讨HPA-1—16基因多态性分布与血小板输注无效相关性。方法应用PCR-SSP法对上海地区268名汉族人群行HPA-1—16基因检测;应用ELISA法对49名反复输血的恶性血液病患者行血小板抗-HLA-Ⅰ与抗-HPA筛查试验。结果上海地区汉族人群HPA-1—16系统中,HPA-1—6,15系统等位基因频率1a=0.9889,1b=0.0111,2a=0.8881,2b=0.1119,3a=0.5989,3b=0.4011,4a=0.9963,4b=0.0037,5a=0.9907,5b=0.0093,6a=0.9832,6b=0.0168,15a=0.6418,15b=0.3582,均呈多态性分布;其余HPA-7—14,16系统等位基因均呈单线性分布。HPA-1,2,4—6,15系统主要以aa纯合子基因型频率分别为0.9776,0.7799,0.9925,0.9813,0.9664,0.4328。在HPA-2、3、15系统中出现bb纯合子基因型,其频率均为0.0037,0.1530,0.1492外,其余系统均未出现bb纯合子基因型。另外,在HPA-1—6,15系统中出现ab杂合子基因型,其频率分别为0.0224,0.2164,0.4963,0.0075,0.0187,0.0336,0.4180,以HPA-3杂合度最高,其次次序为HPA-15、HPA-2。在随机输血中,HPA不合发生率以HPA-3为最高(0.3650),其次分别为HPA-15(0.3541)、HPA-2(0.1790)。49名反复输血的恶性血液病患者中,有61.22%(30名)输血后相继出现抗-HLA-Ⅰ,而始终未检出抗-HPA。结论上海地区汉族人群血小板输注无效的主要原因是抗-HLA-Ⅰ所致;只需检测供者与受(患)者的HPA-2、-3、-15基因相合,就可基本达到血小板匹配性输注。     

复合PCR鉴别葡萄球菌及其多重耐药基因

目的　建立既可鉴别金黄色葡萄球菌又可同时检测其耐药基因的分子诊断方法。方法　对应于femB、mecA、ileS基因的 3对引物与快速提取的单菌落模板DNA进行单管同步扩增 ,电泳观察PCR片段 ;mecA、ileS耐药基因扩增结果分别与苯唑西林、莫匹罗星药敏试验对比 ,分析菌株的耐药性。结果　检测femB基因可快速特异性地筛选出金黄色葡萄球菌 ,mecA基因的检出与常规药敏试验鉴定耐甲氧西林葡萄球菌 (MRS)的结果基本一致 ,而拥有ileS基因的全部葡萄球菌分离株对莫匹罗星耐药。结论　复合PCR可快速敏感地从葡萄球菌中区分金黄色葡萄菌 ,并同时检出MRSA和耐莫匹罗星的多重耐药菌株。