双重实时荧光PCR检测肠出血型大肠埃希菌stx1和stx2基因方法的建立

目的建立一种检测肠出血型大肠埃希菌（EHEC）产毒基因stx1和stx2的TaqMan双重实时荧光PCR法。方法根据GenBank公布的EHECstx1和stx2基因序列进行比对后设计相应的PCR引物和TaqMan探针,探针报告基团分别使用FAM和HEX标记,淬灭基团使用BHQ1标记。建立双重实时荧光PCR反应体系并对反应条件进行优化,对方法的灵敏度、特异性及其重复性进行分析。结果建立了EHEC双重实时荧光PCR检测方法。菌液浓度为102～108cuf/ml时,均可观察到扩增曲线,且扩增曲线平滑;检测30份模拟临床样品,均检测到stx1和stx2基因,且均培养出EHEC,该方法灵敏度为100%;3次重复测定108、105、102cfu/ml3个高、中、低浓度的菌液DNA模板,得到其扩增曲线,计算各浓度的Ct值标准差及变异系数,均小于5%,在误差允许范围内。结论建立的双重实时荧光PCR法可快速、灵敏、特异、有效地检出EHEC。     

肾移植后BK病毒相关性肾病的早期诊断

背景：联合检测对于早期诊断BK病毒相关性肾病有着重要意义,目前研究较少。 目的：探讨肾移植后联合检测诊断BK病毒相关性肾病的方法,并初步分析其临床应用价值。 方法：留取84例肾移植患者的尿液和外周血标本,进行BK病毒感染负荷的实时荧光定量PCR和尿液巴氏染色检测,同时进行移植肾病理穿刺病理组织学及免疫组化检测,用于早期诊断BK病毒相关性肾病。 结果与结论：2例患者诊断为BK病毒相关性肾病,占移植肾活检病例的2.5%。2例患者证实BK病毒相关性肾病后予以免疫抑制剂减量,肾功能明显好转。结果显示以上方法联合检测可提高BK病毒相关性肾病的诊断率,简便、可靠、准确,为进一步研究BK病毒感染与肾移植后移植物丢失的关系奠定了基础。     

马尔堡、埃博拉病毒双重荧光定量PCR检测方法的建立

目的 建立一种快速、敏感、特异的双重实时荧光定量PCR方法,可同时检测马尔堡病毒和埃博拉病毒.方法 通过序列比对挑选出两种病毒基因组中高度保守的序列,分别设计引物及Taqman探针,两条探针分别标记FAM和Texas Red荧光报告基因,建立双重实时荧光定量PCR反应体系.结果 双重荧光定量PCR方法检测两种病毒阳性标准品的灵敏度分别为30.5拷贝/μl和28.6拷贝/μl,通过检测日本脑炎病毒、黄热病毒、登革热病毒无交叉反应,有较好的灵敏度和特异性.结论 建立了马尔堡、埃博拉病毒双重荧光定量PCR检测方法,实现了两种病毒同时实时定量检测,在传染病防控领域有较好的应用前景.     

发热伴血小板减少综合征布尼亚病毒实时荧光RT-PCR检测方法的建立

目的 建立新型发热伴血小板减少综合征布尼亚病毒的TaqMan探针实时荧光RTPCR检测方法并进行评估,为发热伴血小板减少综合征监测中新型布尼亚病毒感染的排查提供实验室检测依据.方法 利用新型布尼亚病毒S片段基因的特异性序列设计引物和探针,探针5'端标记FAM,3'端标记TAMRA,优化反应体系与反应条件,并对不同浓度含...     

基因芯片法特异性检测丙型肝炎病毒的基因分型

目的：采用基因芯片特异性检测血清中丙型肝炎病毒（HCV）并进行基因分型。方法：设计HCV基因型特异探针，将其固定在玻璃片上制成微阵列芯片。阳性组血清60份，阴性组血清15份，乙型肝炎血清5份（抗HCV阴性）。经核酸提取，多聚酶链式反应（PCR）扩增，与芯片上的探针杂交，最后分析结果并与测序分型结果比较。结果：阳性组血清全部检测到HCV-RNA，均有基因芯片分型结果。基因芯片分型结果与测序分型结果一致者56例。阴性组血清HCV-RNA全部阴性。乙型肝炎血清全部阴性。结论：基因芯片可准确对HCV感染血清做定性检测并同时检测HCV基因型，简便快捷，特异性好，并且不需荧光标记和昂贵的荧光扫描仪器，与乙型肝炎血清无交叉反应。可替代基因测序分型，适于临床大量样品的检测。     

PCR基因芯片上荧光PCR反应的研究

目的探讨能否在基因芯片上进行不同的荧光掺入PCR反应并根据基因芯片上荧光的变化判断基因的变异.方法利用健康外周血,正常脐血DNA,X连锁遗传性铁粒幼细胞贫血(XLSA)家系成员6人外周血DNA做PCR-SSCP,并测序确定.设计检测此点突变的Taqman探针进行荧光PCR反应,在芯片上进行同样的反应,与上述反应结果进行对照.利用4步位点特异PCR结合SYBR荧光染料初筛HLAA2,并在芯片上进行同样的反应,与上述反应结果进行对照.结果PCR-SSCP分析与测序确定X连锁遗传性铁幼粒细胞贫血家系两患者的ALAS2基因第5外显子有G514A点突变,母亲、外祖母为杂合子携带者.设计Taqman探针时此家系中六位成员DNA与二份正常男性脐血DNA检测与预期结果相符.将上述结果中同样的样本移入芯片进行PCR反应,结果与常规荧光PCR反应的结果一致.SYBR荧光染料PCR反应与芯片上SYBR荧光染料PCR反应的结果与预期完全相符.结论利用TaqMan探针与SYBR Green荧光染料可以在PCR基因芯片上顺利进行PCR反应,根据基因芯片上荧光的变化检测出点突变与单核苷酸多态性.为基因芯片的临床应用打下了基础.     

实时荧光定量PCR在铁路运输动物携带甲型流感病毒检测中的应用

目的探讨应用实时荧光PCR对铁路运输动物携带甲型流感病毒进行快速检测的可行性,为制定铁路系统有效的流感监测方案奠定基础。方法采集死禽咽喉部和泄殖腔拭子,活体动物用拭子涂抹泄殖腔外周,粪便和毛用拭子涂抹表面。依据《全国流感监测实施方案》、甲型流感病毒核酸检测试剂盒（PCR-荧光探针法）说明书以及ABI7500F型实时荧光定量PCR的SDS软件操作。结果共采集广州中铁快运货场动物样本250份,检出甲型流感病毒核酸阳性11份,阳性率4.4%,其中以鸽类样本的阳性检出率最高,鹅次之,鸭最低。结论实时荧光PCR检测方法适合铁路运输的实际要求,结合进一步病毒分型和病毒基因测序,对掌握病毒流行趋势,做好预防控制工作具有重要意义。     

肝螺杆菌TaqMan MGB探针实时荧光定量PCR快速检测方法的建立及应用研究

目的 建立特异、敏感、快速检测肝螺杆菌的TaqMan MGB探针实时荧光定量PCR方法.方法 针对肝螺杆菌flaB 基因的保守区设计特异性引物和探针,建立肝螺杆菌TaqMan MGB探针实时荧光定最PCR方检测方法,验证方法的特异性、敏感性和稳定性.对2008-2011年期间采集的1081份临床样本中的肝螺杆菌进行检测,同时进行分离培养和常规PCR检测.结果 建立的TaqMan MGB探针实时荧光定量PCR方法对肝螺杆菌的检测具有高度的特异性,对幽门螺杆菌、空肠弯曲菌、泰泽氏菌、侵肺巴斯德氏菌、大肠埃希菌、铜绿假单胞菌均无交叉反应,检测的灵敏度达8.3拷贝.标准曲线显示各浓度范围内具有良好的线性关系,相关系数为0.999,斜率为-3.227,TaqManMGB探针实时荧光定量PCR效率为100％.对1081份临床样本进行检测,TaqMan MGB探针实时荧光定量PCR和常规PCR均能检出86份肝螺杆菌阳性样本,而细菌分离培养则仅检出4份阳性.结果显示,建立的TaqMan MGB探针实时荧光定量PCR方法比细菌分离培养方法更敏感,能够直接从临床样本中检出肝螺杆菌DNA,检测时间仅为2h.结论 研究建立的TaqMan MGB探针实时荧光定量PCR方法具有可靠、特异、敏感的特点,适用于肝螺杆菌的快速检测.     

荧光定量聚合酶链反应检测乙型肝炎病毒DNA

目的 建立检测HBV病毒DNA的荧光定量PCR法（FQ-PCR），并与运用常规凝胶电泳技术观察特异扩增带检测的结果加以比较。方法 合成扩增HBV DNA314bp特异保守序列的1对引物及1条带2个荧光基团的寡核苷酸探针。用PE-5700型定量PCR仪完成PCR反应及产物的荧光定量检测。同是PCR产物经琼脂糖凝胶电泳，EB染色，UVP（凝胶成像仪）检出有314bp带者为阳性或弱阳性。结果 建立了检测HBVDNA的荧光定量PCR技术，用已知HBV阳性模板不同拷贝数的标准溶液测得标准曲线Ct，原始拷贝数在10^5/ml以上者为阳性，用定量及定性PCR2种方法检测698例血清标本的结果表明：用FQ-PCR技术共检测出204例为阳性，阳性率29.2%；用定量及定性PCR2种方法检测698例血清标本的结果表明：用FQ-PCR技术共检测出204例为阳性，阳性率29.2%；用定性PCR观察到193例有阳性特异带，阳性检出率为27.65%。没有发现用定性PCR检测为阳性而用FQ-PCR检测为阴性者。结论 FQ-PCR检测HBVDNA较普通定性PCR技术具有操作简便，灵敏度更高、减少发生污染可导致假阳性结果的可能性及自动化程度高等优点，值得在临床检验中推广应用。     

通用型病毒检测芯片在三种人类致病病毒属检测中的应用

目的 研究制备对人类有致病作用的38个属的病毒寡核苷酸（oligo）基因芯片对三种人类致病病毒的检测能力。方法 应用生物信息学软件设计70-mer oligo探针，固定于玻片载体制备成基因芯片。以痘苗病毒天坛株、甲肝病毒、乙肝病毒作为检测样本，提取病毒核酸，经随机引物扩增、荧光标记，用于芯片杂交，清洗和干燥后对芯片进行扫描和数据分析。结果 芯片上oligo探针能与相应病毒的PCR扩增样品杂交，呈现阳性荧光信号。结论 建立的病毒寡核苷酸基因芯片能够检出和区分三种检测病毒，为进行未知病毒的基因芯片筛查方法的建立奠定了基础。     

Detection and quantitation of BK virus DNA by real-time polymerase chain reaction in the LT-ag gene in adult renal transplant recipients

Determination of polyomavirus BK (BKV) load in urine and plasma has been advocated for monitoring adult renal transplant recipients suffering from BKV-related nephropathy. An "in-house" real-time quantitative PCR assay was developed using the BKV-1/BKV-3 primers set in the large tumor antigen (LT-ag) region to quantitate BK virus loads in plasma and urine in renal transplant patients. This assay was adapted to routine virology laboratory by evaluating two extraction procedures of nucleic acids from urine and plasma, one manual and the other using an automatic extractor, and by evaluating the Light Cycler versus Taqman apparatus. Both the manual and automatic extraction procedures and real-time PCR apparatus were equivalent. The Light Cycler and Taqman instruments allow similarly rapid, accurate, reproducible and specific quantitative detection of the three major BKV subtypes, with a detection limit of 10 BKV DNA copies/ml, and a range from 10(0) to 10(7) copies/ml. Of 855 renal transplant patients, 128 (15%) had BKV DNA in both plasma and urine samples with a mean viral load of 5.1 log/ml in plasma and 6.8 log/ml in urine and in 5 (4%) BKV-associated tubulo-interstitial nephropathy; 332 (39%) BKV DNA was found only in the urine, not in the plasma, without further development of nephropathy and 395 patients had no BKV in plasma and urine. These observations emphasize the usefulness of real-time PCR to assess the BKV load by routine testing, and confirm the need to combine both plasma and urine determinations of the BKV DNA load in order to identify renal transplant patient at high risk for BKV-associated nephropathy.     

Genotypes of human polyomaviruses in urine samples of pregnant women in Taiwan

The viral DNA of human polyomaviruses JC virus (JCV) and BK virus (BKV) was detected by the polymerase chain reaction (PCR) in urine samples from 31 pregnant women in Taiwan. A pair of appropriate primers amplified both JCV and BKV DNA of the regulatory region simultaneously in PCR. An oligonucleotide probe homologous to both JCV and BKV regulatory region was used subsequently to detect the viral DNA by Southern blotting after PCR amplification. Approximately 36% of the examined urine samples were human polyomavirus positive. The genotypes of JCV and BKV were determined by DNA sequencing of their regulatory regions. Besides CY archetype, a new strain (Taiwan-1) of JCV with a pentanucleotide (GGGAA) deletion and a new strain (Taichung-1) of BKV with two nucleotide alterations within the regulatory region were found in the urine samples. Eight of the examined samples were JCV infected, one was BKV infected, and two were JCV and BKV mix-infected. The JCV positive individuals were infected by CY archetype and Taiwan-1 strain equally. However, Taichung-1 strain was the only BKV strain found in the BKV positive individuals. © 1996 Wiley-Liss, Inc.     

Detection of BK virus and JC virus in urine and brain tissue by the polymerase chain reaction.

DNAs of the human polyomaviruses BK virus (BKV) and JC virus (JCV) were amplified by the polymerase chain reaction (PCR) by using a single pair of 20-base oligonucleotide primers that were complementary to the same regions of both viruses. The sequences flanked by the primers were unique for each virus and could be differentiated by hybridization with 40-base, 32P-labeled oligonucleotide probes or by cleavage with BamHI. The DNA fragments resulting from amplification of BKV and JCV were 176 and 173 nucleotide pairs, respectively. The sensitivities of PCR for amplification of cloned BKV and JCV DNAs were 10 and 100 copies, respectively. Hybridization with the oligonucleotide probes was specific for each virus. A total of 57 urine samples from three groups of subjects were processed by DNA extraction or boiling and were tested by PCR. Urine samples collected from immunosuppressed patients (n = 11) and previously documented to be positive for BKV, JCV, or both were positive by PCR. Ten percent of urine samples from healthy adults (n = 30) that were previously negative for BKV and JCV were positive for one or both viruses by PCR. Urine samples (n = 16) from four seronegative bone marrow transplant recipients were uniformly negative for BKV. JCV was detected in deparaffinized brain tissue from a patient with progressive multifocal leukoencephalopathy. Specific diagnosis of virus in clinical specimens could be made within 1 day of receipt of the specimens. The PCR method is attractive for use in diagnosing polyomavirus infections because of its sensitivity, specificity, and rapid turnaround time.     

New commercially available PCR and microplate hybridization assay for detection and differentiation of human polyomaviruses JC and BK in cerebrospinal fluid, serum, and urine samples

JC and BK human polyomaviruses (family Polyomaviridae) may cause severe neurological or urinary tract pathologies in immunocompromised hosts. In the present study, we evaluated a new commercially available PCR and microplate colorimetric hybridization assay for the standardized differential detection of JC virus (JCV) and BK virus (BKV) genomes in clinical samples. This JC/BK Consensus test was first evaluated by testing serial dilutions of JCV or BKV plasmid DNA standards and was then compared with an in-house reference PCR assay for the detection of JC and BK virus genomes in 70 cerebrospinal fluid (CSF) samples of patients with neurological disorders and in 75 serum or plasma samples and 125 urine samples of renal graft recipients. This new test allowed a limit of detection of 10 copies and 1 copy of JC and BK virus genomes, respectively, and was able to differentiate various levels of JCV, BKV, and mixed JCV and BKV DNA genomes in a single reaction tube. Our results showed 100% specificity and sensitivity for the JC/BK Consensus test with CSF samples. With serum or plasma samples, this test had a sensitivity and a specificity of 100% for both JCV and mixed JCV and BKV DNA detection and a sensitivity and a specificity of 100 and 97.8% for BKV DNA detection, respectively. With urine samples, the sensitivity and specificity were 100 and 96.6%, respectively, for JCV DNA detection; 100 and 89.4%, respectively, for BKV DNA detection; and 44.4 and 100%, respectively, for mixed JCV and BKV DNA detection. In conclusion, our data indicate that this new test, the JC/BK Consensus test, is valuable for the sensitive and specific differential detection of single JCV and BKV infections in CSF, serum or plasma, and urine samples. The use of this reliable PCR assay would improve the routine virological diagnosis as well as the clinical care of immunocompromised patients with polyomavirus-related pathologies.     

Evaluation of a BK virus real-time PCR assay designed using novel bioinformatics technology

Monitoring of active polyomavirus BK (BKV) infections by quantitative real-time PCR is becoming a progressively more routine practice in the care of renal transplant patients due to the potential for these infections to injure transplanted kidneys. Quantitative BKV results from a previously validated, laboratory-developed real-time PCR assay based on commercially available MGB Alert® reagents were compared to results obtained from the same urine and plasma specimens using a commercially designed real-time PCR assay by IntelligentMDx. When compared qualitatively, the two assays performed identically with the exception of one urine specimen in which BKV DNA was detected near the lower limit of quantification by the MGB Alert® assay. A quantitative comparison of the results showed an average 0.55 log₁₀ copies/mL difference between the two assays. These findings suggest that despite small differences, the IntelligentMDx assay could be adopted for clinical BKV monitoring of renal transplant patients.     

Specific Detection of Human BK Polyomavirus in Urine Samples of Immunocompromised Patients

A semiquantitative PCR assay for the detection of BK virus in urine was developed using primers for BK virus that specifically amplified BK but not JC virus. DNA was extracted from urine through treatment with proteinase K followed by DNA precipitation with sodium acetate. Semiquantitation was achieved by amplifying serial dilutions (1:1, 1:10, 1:100, and 1:1,000) of the urine specimens. Each assay included both positive (stock BK virus and previously positive patient urine) and negative (no template) controls. A urine sample was interpreted as positive if any of the serial dilutions showed amplification of the DNA fragment of the expected size. For some patient-derived samples, amplification of the expected-size fragment was achieved with a dilute template whereas no amplification was achieved with a concentrated template. This was attributed to interfering substances in the urine. PCR results were compared with urine cytology and shown to be more sensitive. Validation studies were performed at the University of Nebraska Medical Center, utilizing a separate qualitative PCR assay that detects both BK and JC virus and distinguishes between them by restriction enzyme digestion patterns. Of 46 urine samples analyzed using both methods, 22 were positive by both assays, 18 were negative by both assays, 5 were positive only by the Nebraska method, and 1 was positive only by our method. In comparison with the Nebraska PCR, our PCR assay had a sensitivity of 81% and specificity of 95%. For twenty-one (43%) of 49 immunocompromised patients, tests were postive when specimens were submitted because of clinical suspicion of BK virus infection.     

Specific detection of human BK polyomavirus in urine samples of immunocompromised patients

A semiquantitative PCR assay for the detection of BK virus in urine was developed using primers for BK virus that specifically amplified BK but not JC virus. DNA was extracted from urine through treatment with proteinase K followed by DNA precipitation with sodium acetate. Semiquantitation was achieved by amplifying serial dilutions (1:1, 1:10, 1:100, and 1:1,000) of the urine specimens. Each assay included both positive (stock BK virus and previously positive patient urine) and negative (no template) controls. A urine sample was interpreted as positive if any of the serial dilutions showed amplification of the DNA fragment of the expected size. For some patient-derived samples, amplification of the expected-size fragment was achieved with a dilute template whereas no amplification was achieved with a concentrated template. This was attributed to interfering substances in the urine. PCR results were compared with urine cytology and shown to be more sensitive. Validation studies were performed at the University of Nebraska Medical Center, utilizing a separate qualitative PCR assay that detects both BK and JC virus and distinguishes between them by restriction enzyme digestion patterns. Of 46 urine samples analyzed using both methods, 22 were positive by both assays, 18 were negative by both assays, 5 were positive only by the Nebraska method, and 1 was positive only by our method. In comparison with the Nebraska PCR, our PCR assay had a sensitivity of 81% and specificity of 95%. For twenty-one (43%) of 49 immunocompromised patients, tests were positive when specimens were submitted because of clinical suspicion of BK virus infection.     

The use of unprocessed urine samples for detecting and monitoring BK viruses in renal transplant recipients by a quantitative real-time PCR assay

Results of quantitative BK viral load using real-time quantitative PCR (rt-QPCR) were compared using two types of samples, extracted urine DNA and unprocessed urine. An excellent correlation was observed in quantitative viral load between unprocessed urine and extracted urine DNA samples. (R2=0.96, p<0.001). Compared to extracted urine DNA when a small sample volume of unprocessed urine was used (2 microl per PCR reaction), 100% concordance is detection of BKV DNA was observed in 124 samples (106 positive and 18 negative) collected from renal transplant recipient (RTR). There was no significant difference in the quantitative BK viral load (log10 copies/ml) detected in extracted urine DNA (median=7.82) compared to unprocessed urine (median=7.17). Urine pH in the range of 5.2-7.1 and specimen freezing had no effect on the rt-QPCR reaction. The partial inhibition of the rt-QPCR reaction observed when 5 microl sample volume of unprocessed urine was used was markedly reduced at a sample volume of 2 microl. Using unprocessed urine for rt-QPCR detection of BK viral load is cost-saving while maintaining the sensitivity and accuracy associated with the use of extracted urine DNA, making a clinical BKV surveillance strategy in RTR based on urinary sample screening using rt-QPCR as the first line test more feasible.     

Reevaluating and optimizing polyomavirus BK and JC real-time PCR assays to detect rare sequence polymorphisms

PCR-based molecular assays have a central role in polyomavirus diagnostics. To assure optimal performance, target sequences should be regularly updated according to newly available sequences. The aim of this study was to review our in-house polyomavirus BK (BKV) and JC (JCV) real-time PCR assays. Database analysis revealed variations in the BKV target region which might affect the assay performance, while no significant changes were found in the JCV target region. We compared two degenerate versions of our BKV primers which accommodated at least 95% of all published genetic variants. Dilutions of cloned viral genomic DNA and probit analysis indicated an analytical sensitivity of the updated BKV assay of 4.15 copies/reaction and that of the JCV assay was 3.37 copies/reaction. The specificity was assessed by testing JCV- and BKV-positive samples that showed no cross-reactivity. The performance of the original and updated BKV assay was compared in 101 urine and 200 plasma samples submitted to our routine diagnostic laboratory revealed similar quantitative results. We conclude that our JCV and updated BKV real-time PCR assays are robust and detect rare variants possibly encountered in the clinical routine.