Detection of alpha-thalassemia-1 Southeast Asian type using real-time gap-PCR with SYBR Green1 and high resolution melting analysis

Alpha-thalassemia-1 Southeast Asian (SEA) type is the most common genetic disorder in the Asian population. Couples who are both carriers have a 25% chance of conceiving Bart's hydrops fetalis. Therefore, results from carrier screening and prenatal diagnosis frequently need to be available rapidly. A rapid technique for diagnosis of alpha-thalassemia-1 SEA type was implemented. The technique used is based on real-time gap-PCR and high resolution melting (HRM) analysis of the amplified fragment using the Rotor-Gene 6000. The DNA samples used for amplification were obtained from whole blood, cord blood, and chorionic villus sampling (CVS). With this method, the alpha-thalassemia-1 SEA allele can be easily distinguished from wild type alpha-globin gene allele. The real-time gap-PCR and HRM analysis offers additional benefits including minimal labor, rapid turnaround time, and a decreased risk of PCR carryover contamination. It is cost-effective and safe, does not require fluorescently labeled probe and hazardous chemicals. Moreover, it is accurate showing 100% concordance with conventional gap-PCR analysis.     

TaqMan MGB探针实时聚合酶链反应检测登革病毒

目的建立一种敏感、特异、重复性好的登革病毒(DengueVirus,DV)鉴定方法。方法根据DV3’端非编码区基因保守序列,设计一套特异性引物和TaqManMGB探针。利用1998~2004年收集的26份登革热病人临床血清标本,15例乙脑病人血清,DV4个血清型标准毒株及23株1978~1997年DV地方流行株和相关西尼罗病毒、日本脑炎病毒、麻疹病毒、基孔肯亚病毒等毒株,同时用克隆了1型DV基因组3’端非编码区序列片段的质粒DNA作为阳性对照,检测所建立方法的特异性、敏感性。结果所建立方法的最低检测限约为每反应5个基因拷贝。用该方法检测4个血清型DV标准毒株、23株DV地方流行株和26例分离到DV的阳性血清标本,检出率为100%;用该方法检测15例乙脑病人血清、10例麻疹病人血清和西尼罗病毒、日本脑炎病毒、麻疹病毒、基孔肯亚病毒,结果均为阴性。结论新建的TaqManMGB探针检测DV方法是实验室早期诊断登革热较理想的方法。     

SYBR Green Ⅰ实时荧光定量PCR检测BMI-1 mRNA方法的建立

目的 建立SYBR Green Ⅰ实时荧光PCR定量检测人类BMI-1 mRNA的方法.方法 以含有目的基因BMI-1 cDNA的质粒pEGFP-BMI-1为阳性模板,进行纯化和定量及系列稀释,应用SYBR Green Ⅰ实时荧光定量PCR检测BMI-1,建立标准曲线,熔解曲线分析产物的特异性.结果 该法检测的线性范围为6.4×101～6.4×107拷贝,相关系数r为-1.00,6.4×103拷贝/反应的标本批内变异系数(CV)和日间CV分别为1.6%和2.8%.熔解曲线分析显示单一的峰,Tm为(80.40±0.18)℃.结论 SYBR Green Ⅰ实时荧光PCR定量检测BMI-1 mRNA是快速有效、灵敏度高、特异性好的方法,可为BMI-Ⅰ的进一步研究奠定基础.     

FY*X real-time polymerase chain reaction with melting curve analysis associated with a complete one-step real-time FY genotyping

BACKGROUND AND OBJECTIVES: The Duffy (FY) blood group system is controlled by four major alleles: FY*A and FY*B, the Caucasian common alleles, encoding Fy(a) and Fy(b) antigens; FY*X allele responsible for a poorly expressed Fy(b) antigen, and FY*Fy a silent predominant allele among Black population. Despite the recent development of a real-time fluorescent polymerase chain reaction (PCR) method for FY genotyping FY*X genotyping has not been described by this method. This study focused on the real-time FY*X genotyping development associated with a complete, one-step real-time FY genotyping, based on fluorescence resonance energy transfer (FRET) technology. MATERIALS AND METHODS: Seventy-two blood samples from Fy(a+b-) Caucasian blood donors were studied by real-time PCR only. Forty-seven Caucasian and Black individual blood samples, referred to our laboratory, were studied by PCR-RFLP and real-time PCR. For each individual, the result of the genotype was compared to the known phenotype. RESULTS: The FY*X allele frequency calculated in an Fy(a+b-) Caucasian blood donors population was 0.014. With the Caucasian and Black patient samples we found a complete correlation between PCR-RFLP and the real-time PCR method whatever the alleles combination tested. When the known phenotype was not correlated to FY*X genotype, the presence of the Fy(b) antigen was always confirmed by adsorption-elution. CONCLUSION: The real-time technology method is rapid and accurate for FY genotyping. From now, we are able to detect the FY*X allele in all the alleles combinations studied. Regarding its significant frequency, the detection of the FY*X allele is useful for the correct typing of blood donors and recipients considering the therapeutic use of blood units and the preparation of test red blood cells for antibody screening.     

Parvovirus B19 genotypes 1 and 2 detection with real-time polymerase chain reaction assays

BACKGROUND AND OBJECTIVES: Parvovirus B19 (B19V) DNA screening has been introduced to comply with European regulations for certain plasma products. Current commercial and some in-house B19V DNA assays fail to detect or under-quantify the recently identified genotypes 2 and 3. In this report, we describe 2-year experience with B19V DNA screening using the commercial assay from Roche (detecting only genotype 1) combined with an in-house assay (detecting genotypes 1, 2 and 3). This dual testing approach enables the identification of molecular variants of B19V. MATERIALS AND METHODS: Between 2005 and 2007, approximately 2.6 million plasma donations were screened for B19V DNA loads exceeding 10(6) IU/ml using the Roche and the in-house real-time polymerase chain reaction assay. RESULTS: A total of 232 plasma units were identified with B19V DNA loads above 10(6) IU/ml. Concordant results were observed for the majority of B19V positive samples; however, three of these showed discrepant results between the two assay systems. One was a B19V genotype 2 strain not detected by the Roche assay; another was a B19V genotype 1 strain with a mismatch in the 3'-end of the reverse primer and therefore under-quantified by the Roche assay; and the third one was also a B19V genotype 1 strain that gave an unusual amplification plot in the in-house assay due to a mismatch in the probe-binding site. CONCLUSIONS: New, high viral load, B19V genotypes 2 and 3 infections are rare in blood donors tested by Sanquin. One case was found while testing 2.6 million donations. The prevalence of B19V genotype 1 variants not detected by commercial or in-house assays might be in the same range or even higher than the prevalence of B19V genotype 2 viruses, which remain undetected.     

应用Taq Man荧光PCR定性定量检测炭疽芽孢杆菌

目的　发展一种快速、敏感、特异的检测炭疽芽孢杆菌的方法。方法　应用基于TaqMan荧光探针的实时(real time)PCR技术,针对致病炭疽毒株的两个质粒pXO1、pXO2上的pagA ,cap基因和染色体上的ropB基因设计引物和探针定性、定量检测炭疽芽孢杆菌。构造并应用上述探针的外标对照及pagA的内标对照,采用多重荧光定量PCR技术建立荧光定量方法并发现假阴性;采用UDG抗污染和ROX矫正背景噪音,提高检验能力;用该方法检测炭疽芽孢杆菌疫苗株感染的动物脾脏标本,盲法评价该方法在实际工作的应用。结论　该方法能特异、灵敏、高效地检测炭疽芽孢杆菌,该方法的推广和应用对有效防范炭疽生物恐怖袭击、提高突发事件应对能力、快速诊断具有重要意义。     

Storage duration and polymerase chain reaction detection of Plasmodium falciparum from blood spots on filter paper

To evaluate the effect of long-term storage of sample filters on the sensitivity of polymerase chain reaction (PCR) detection of malaria, 252 blood spots from patients with microscopically confirmed Plasmodium falciparum malaria were analyzed and stratified by storage duration. The spots were collected between 1996 and 2000 on filter paper and stored at room temperature. A Chelex-based method was used to extract the DNA. Unexpectedly, after the first purification, the sensitivity of the PCR from recently stored samples was low and showed progressively increased with time storage (P = 0.003, by chi-square test for linear trend). This suggested that PCR inhibitors were easier to dissolve from the more recent blood spots (< 4 years old) than from blood spots > or = 4 years old, thus leading to a time-dependent increase in PCR sensitivity. However, if DNA was purified again (when the first PCR result was negative), the cumulative sensitivity was not influenced by storage duration. This indicated that length of storage is not a critical issue providing purification is sufficient.     

Rapid detection of the major Mediterranean beta-thalassaemia mutations by real-time polymerase chain reaction using fluorophore-labelled hybridization probes

We present a new method to detect the major beta-thalassaemia mutations of the Mediterranean countries (IVS I-1, IVS I-6, IVS I-110, CD-37 and CD-39). The procedure is based upon real-time polymerase chain reaction (PCR), using specific fluorescently labelled hybridization probes. The melting curves for each of the specific probes obtained after PCR enabled the identification of different alleles. Genotyping of 71 patients with thalassaemia and 20 controls without thalassaemia by the established method and conventional allele-specific restriction enzyme analysis produced identical results. The established method is a robust, fast and straightforward assay that allows detection of the major Mediterranean beta-thalassaemia mutations simultaneously in less than 60 min.     

滤纸片干血斑用于HIV-1 DNA检测的评价

目的 探索滤纸固定艾滋病病毒Ⅰ型核酸(HIV-1 DNA)的稳定性、均一性,评价滤纸片干血斑HIV-1 DNA检测方法的敏感性、特异性.方法 套式PCR扩增HIV-1的env、gag、pol三区.不同环境条件下保存的滤纸片干血斑,分别在不同的时间段检测;滤纸片干血斑的中心及圆斑的上下左右4个边缘,随机打取5个2.5mm圆片用于检测.采自新疆现场的94份阴性样本和90份阳性样本,分别制成滤纸片干血斑样本和全血样本,比较两者的HIV-1DNA检测结果.结果 滤纸片干血斑-20℃或4℃放置1年或室温放置3个月,或在37℃放置两天或冻融2次,实验的敏感性未见下降;37℃放置3天或冻融3次,已经有样本不能检出.滤纸干血斑不同位点随机打取圆片扩增,4个样本中,中点均阳性,但在上点、左点、右点分别出现一份阴性结果.滤纸干血斑法和全血法检测HIV-1前病毒DNA比较,敏感性是96.1%,特异性是98.9%.结论 滤纸干血斑在室温条件下运输是可行的,推荐尽量靠近滤纸干血斑的中间位点打取圆片扩增;该方法操作简便、经济、敏感、特异.     

医院临床标本中阴沟肠杆菌遗传多样性分析及应用TaqMan荧光定量PCR方法快速检测阴沟肠杆菌的研究

目的 调查医院临床标本中阴沟肠杆菌基因群的构成比例,并建立TaqMan荧光定量PCR方法对阴沟肠杆菌进行特异、灵敏、快速检测.方法 通过hsp60基因分型分析临床标本中基因群的构成,并以外膜蛋白基因(ompⅩ)为靶基因设计引物及FAM探针,建立TaqMan荧光定量PCR方法对阴沟肠杆菌基因群检测,并评价该方法的特异性、灵敏性和稳定性.结果 237株临床标本共归为10个基因群.其中群Ⅲ,Ⅵ和Ⅷ菌株数量最多,占总菌株数的71％;群Ⅰ占11％;其他6个群总共占18％.无基因群Ⅶ,Ⅹ和Ⅻ.TaqMan荧光定量PCR方法能对阴沟肠杆菌不同基因群进行特异检测:对十个基因群和群Ⅰ的质粒标准品的检测下限分别为36拷贝/μL和21拷贝/μL,对粪便模拟标本的检测下限为104个菌落形成单位/μL;TaqMan荧光定量PCR方法对质粒标准品和粪便模拟标本检测的扩增曲线良好;结论 医院临床标本中可以检测到十个基因群,具有遗传多样性的特征.本研究建立的TaqMan荧光PCR方法特异性好、灵敏度高,能够用于阴沟肠杆菌的快速检测.     

应用SYBR Green I荧光定量RT-PCR测定糖尿病大鼠肾皮质TGF-β1表达水平

SYBR GreenI荧光定量RTPCR法检测正常、糖尿病和牛磺酸治疗组大鼠TGFβ1mRNA/βactinmRNA比值为(7.0±0.8)×10-3,(64.4±8.0)×10-3和(16.7±2.0)×10-3;终点法RTPCR检测比值分别为0.28±0.12,0.58±0.16和0.43±0.10。与终点法RTPCR相比,TGFβ1mRNASYBRGreenI荧光定量RTPCR实时检测法,方便快捷,敏感特异。     

Analysis of real-time SYBR-polymerase chain reaction cycle threshold for diagnosis of the alpha-thalassemia-1 Southeast Asian type deletion: application to carrier screening and prenatal diagnosis of Hb Bart's hydrops fetalis

Without gel electrophoresis and specific probes, the two tubes real-time SYBR-polymerase chain reaction (SYBR-PCR) was setup by using different primer sets: P1/P2 for the detection of wild type alpha-globin gene alleles and P1/P3 for detection of the allele bearing the Southeast Asian (SEA) type (--SEA) deletion. Analyses of the cycle threshold (CT) values obtained by each primer set together with a delta-cycle threshold (DeltaCT) and CT ratio, showed that lower CT values generated by primer sets P1/P2 and P1/P3 were observed in normal and Hb Bart's hydrops fetalis subjects, respectively. In heterozygous subjects the CT values generated by both sets of primers were similar to each other. There was no overlapping of DeltaCT and CT ratio between normal, heterozygous and Hb Bart's hydrops fetalis subjects. Therefore, the two tubes real-time SYBR-PCR could represent a rapid, cost effective, high-throughput assay for screening of carriers and prenatal diagnosis of alpha-thalassemia-1 (alpha-thal-1) with the SEA type (--SEA) deletion.     

Comparison of real-time quantitative polymerase chain reaction with three other assays for quantitation of hepatitis C virus

BACKGROUND AND AIMS: Evaluation of serum levels of hepatitis C virus (HCV) is important for predicting the response to interferon treatment and monitoring its therapeutic efficacy. The aim of this study was to evaluate real-time quantitative polymerase chain reaction (PCR) as a method for the measurement of HCV-RNA. METHODS: The subjects were 50 patients with chronic hepatitis C: 36 with genotype 1b, eight with genotype 2a, and six with genotype 2b. Samples were tested for HCV-RNA by using real-time quantitative PCR with the ABI Prism 7700 sequence detection system, a branched DNA signal amplification assay, and an Amplicor monitor test; and for HCV core protein by using a fluorescent enzyme immunoassay. RESULTS: The detection range of the real-time quantitative PCR was between 10(1)-10(8) copies/mL of HCV-RNA. Hepatitis C virus RNA was detectable in all 50 samples by the use of real-time quantitative PCR, but was undetectable in 14 samples by the use of a branched DNA assay and in two samples by using the Amplicor monitor test; HCV core protein was undetectable in three samples. A significant correlation was found between the results of real-time quantitative PCR and those of the three other assays: branched DNA assay (r = 0.837, P < 0.0001), Amplicor monitor test (r = 0.853, P < 0.0001), and HCV core protein concentrations (r = 0.549, P < 0.0001). CONCLUSIONS: Our results showed that the real-time quantitative PCR was a highly sensitive assay for the measurement of HCV-RNA.     

Clinical trial of quantitative real-time polymerase chain reaction for detection of cytomegalovirus in peripheral blood of allogeneic hematopoietic stem-cell transplant recipients

The preemptive therapy of cytomegalovirus (CMV) reactivation is useful for the prevention of CMV disease in allogeneic hematopoietic stem-cell transplant (HSCT) recipients. We compared results of the pp65 CMV antigenemia test with quantitative touch-down polymerase chain reaction (Q-PCR) on unfractionated whole blood for the detection of CMV reactivation in 51 HSCT recipients. Forty episodes of reactivation in 28 patients were detected by antigenemia and treated by antiviral drugs. Q-PCR detected CMV DNA in 39 (97.5%) of 40 reactivation episodes. False-positive results occurred in 3% of tests, of which 63% were borderline positive. Q-PCR results were positive earlier than antigenemia results in 30 (77%) of 39 episodes detected by antigenemia. Q-PCR remained positive after treatment was discontinued in 14 (36%) of 39 episodes and predicted the return of CMV reactivation in 4 (31%) of 13 episodes. Q-PCR was more sensitive than the antigenemia test and had sufficient specificity for clinical use.