骨髓涂片人微小病毒B19的检测方法

用ＰＣＲ方法检测人骨髓细胞微小病毒Ｂ１９ＤＮＡ。设计了二对特异引物，ＰＣＲ反应后，出现期望６９９ｂｐ及３１５ｂｏ条带。并用Ｂ１９特异探针确认其特异性。套式ＰＣＲ灵敏度可达１ｆｇ。１％ＮＰ－４０法处理样品简便实用。分析５０例选择性临床血液病患者骨髓涂片，发现有明显贫血症状的血液病存在较高的Ｂ１９感染率。     

应用PCR法对临床血标本中人微小病毒B19的检测分析

目的探讨PCR法检测临床血标本中人微小病毒B19(HPV B19)的应用价值。方法根据序列比对结果,在HPV B19核苷酸相对保守区设计引物进行PCR扩增。应用双脱氧链末端终止法对HPV B19阳性PCR产物进行克隆测序。结果应用该法最终检出HPV B19的血清稀释度为10^3。序列比较表明,用本法检出的1株HPV B19阳性标本与标准株(Au株)核苷酸序列同源性为92％。检测肝癌和肝硬化患者血清标本各14例,结果HPV B19阳性分别为8例和5例。结论本法可用于HPV B19的临床诊断和流行病学调查。     

人类微小病毒B19的检测方法及进展

人类微小病毒B19（简称B19）由Cossart等于1975年首先发现。B19是经呼吸道（鼻咽）正常传播，大约有50％的成年人可找到B19抗体。B19病毒可经血液和血液制品传播，并能产生人体免疫反应。2000年，WHO组织14个国家26个实验室完成一项合作研究一建立人类细小病毒B19DNA核酸扩增技术的国际标准．本文就B19病毒的生物学、流行病学、检测方法等研究现状做一综述。     

荧光定量聚合酶链反应技术检测儿童患者人巨细胞病毒结果分析

目的 了解本院儿童患者人巨细胞病毒(HCMV)感染现状.方法 采用实时荧光定量聚合酶链反应(PCR)技术对243例住院疑似患儿进行HCMV-DNA定量检测.结果 HCMV总阳性率为16.05%;男性和女性阳性率分别为17.83%和12.79%;1个月至1岁年龄组(婴儿期)患者最高为17.41%.临床诊断主要以肝损害、腹泻和黄疸为主.结论 加强儿童患者HCMV的检测,有助于临床诊断与治疗方案的选择.     

无偿献血人群人微小病毒B19感染状况调查

目的 在现有血液筛查模式下,对苏州地区无偿献血者感染人微小病毒B19的情况进行调查研究。方法 随机选取2022年9―12月苏州地区无偿献血者893名,用ELISA法进行B19血清学IgG、IgM抗体检测,并对抗体阳性者进行B19 DNA real-time PCR检测。结果 893份标本中,B19抗体总阳性率为20.7%(185/893),IgG、IgM抗体阳性率分别为19.4%(173/893)、1.9%(17/893),2者有差异(P<0.05);男性与女性B19 IgG及IgM抗体阳性率(20.1%,1.5%vs 18.0%,2.6%)均无差异(P>0.05);B19 IgG抗体阳性率随着献血者年龄增长逐渐升高(P<0.05),但IgM抗体阳性率无差异(P>0.05);不同血型人群B19 IgG及IgM抗体阳性率均无差异(P>0.05);重复献血者B19 IgG抗体阳性率高于初次献血者(21.5%vs 15.9%)(P<0.05),IgM抗体阳性率则无差异(P>0.05);在B19抗体阳性标本中,检出B19 DNA阳性3份,阳性率为1.6...     

利用实时荧光定量聚合酶链式反应快速检测人及动物粪便中的沙门菌

目的为了快速检测、鉴定沙门菌属细菌,提高食源性疾病暴发应对能力,本研究建立了针对沙门菌属的实时荧光定量聚合酶链式反应(qPCR)检测方法,并对其特异性、敏感性和检测下限进行评价。方法筛选针对沙门菌的属特异基因,并建立该基因的qPCR检测体系,利用肠道不同种属细菌、不同亚种及血清型沙门菌属细菌、动物及人粪便样本评价该体系的特异度、灵敏度及检测下限。结果获得沙门菌的属特异基因ttrA,建立基于该基因的qPCR检测方法。发现该方法对纯DNA的最低检测下限为2拷贝/反应。对沙门菌属以外的肠道致病菌无扩增,对1 100株不同亚种及血清型的沙门菌的扩增结果均为阳性,对150份沙门菌致腹泻患者的粪便增菌液和210份动物带菌粪便增菌液均检测阳性。结论本研究建立的基于单一基因的沙门菌属快速分子检测方法具有特异度高、灵敏度高的特点,可用于快速筛查、鉴定沙门菌及由其引起的感染性腹泻。     

利用实时荧光定量聚合酶链式反应快速检测鸭沙门菌

目的为快速检测、鉴定鸭沙门菌，避免血清型误判，提高鸭沙门菌暴发应对能力，建立一种实时荧光定量聚合酶链式反应（qPCR）检测方法。方法随机挑选60株鸭沙门菌及238株肠道常见病原菌代表菌株，针对鸭沙门菌血清型特异基因AW58_15605设计引物并建立qPCR检测体系，通过纯菌菌株、模拟粪便样本及临床样本评价该体系的特异度、灵敏度及检测下限。结果60株鸭沙门菌株及临床感染的50份样本扩增结果均为阳性，而对鸭沙门菌血清型以外的其他沙门菌及肠道菌的扩增均为阴性。 对鸭沙门菌纯菌DNA的最低检测下限为8拷贝/反应。 粪便模拟样本检测中，增菌后qPCR检测下限达59.10 cfu/g，明显低于分离培养及增菌前。结论本研究建立的基于特异基因的鸭沙门菌分子检测方法具有可操作性强、特异度高、灵敏度高的特点，建议在应急情况下优先选择qPCR用于沙门菌暴发筛查、鉴别及诊断由其引起的腹泻性疾病。     

南宁地区育龄妇女人类微小病毒B19流行病学调查

目的 了解南宁地区育龄妇女人微小病毒B19(B19)感染状况及特点.方法 用酶联免疫吸附法检测2008～2010年南宁地区育龄妇女血清B19-IgM抗体,比较不良妊娠结局、妊娠和婚检妇女感染率及各年、季度感染情况.结果 10 125例育龄妇女,B19感染率为13.04%,不良妊娠结局组、妊娠组和婚检组感染度分别为17.69%,12.61%和10.82%,三组间感染率差异有统计学意义(P<0.01).各年感染率分别为12.20%,13.18%和13.68%,各年、季度感染率差异无统计学意义(P>0.05).感染组与非感染组不良妊娠结局发生率差异有统计学意义(P<0.01).结论 南宁地区育龄妇女B19感染率较高,应加强育龄妇女B19感染监测.     

Detection of parvovirus B19 by dot-blot and polymerase chain reaction

Our studies compare detection of parvovirus B19 DNA by dot-blot hybridization and by the polymerase chain reaction (PCR). Compared to detection by dot-blot hybridization with a 32P-oligonucleotide probe, the sensitivity of PCR product detection by ethidium bromide fluorescence is not compromised when the size of the amplification target and number of cycles of amplification are properly selected. Our PCR assay simultaneously amplifies two targets in the B19 genome and yields a sensitivity 10 times greater than 32P-labelled oligonucleotide--dot-blot hybridization (dot-blot). In 126 serum samples from cases of suspected parvovirus B19 infection, viral DNA was detected by PCR in 17% (21/126), whereas dot-blot detected B19 DNA in 0.8% (1/126). Parvovirus B19-specific antibodies were detected in 69% (87/126) of the suspected clinical cases by immunoblot. Nineteen of the antibody-positive specimens were DNA positive of which three were IgM-positive/IgG-negative, 11 IgM-positive/IgG-positive and five IgM-negative/IgG-positive. The only dot-blot DNA positive sample was also PCR-DNA positive but was B19 IgM-negative/IgG-negative. Eighty-six B19 antibody-negative, clinically uninfected controls were also negative for B19 DNA by PCR and by dot-blot. These studies confirm the increased sensitivity of gene amplification by PCR for detection of B19 DNA and demonstrate that two targets in B19 DNA can be amplified simultaneously with resultant increased ease of interpretation. Finally, detection of the two B19-specific products by ethidium bromide fluorescence is documented.     

乙型肝炎病毒实时荧光定量PCR检测方法的建立

目的建立一种采用Lightcycler系统进行HBVDNA实时荧光定量PCR的方法,并探讨其临床应用价值。方法针对HBV基因组S区设计一对扩增引物,并通过预实验严格优化反应体系的组成和条件;将T载体与HBVRT区扩增后纯化的产物进行连接反应,然后转染大肠杆菌（DH5a）,经蓝、白斑筛选后挑取阳性菌落,提取质粒,制备外标准品。结果用于制成外标准品的质粒经1：10的缓冲液倍比稀释,制作标准曲线,线性方程为：Y=-3．344X＋37（r2=0．9999）;通过检测已知浓度并经倍比稀释的HBVDNA,表明其最低检测限为5×10^2 IU／mL;拷贝数介于5×10^2～5×10^8 IU／mI。之间的HBVDNA浓度与ct值具有良好的线性关系。结论外标法实时荧光定量PCR是一种定量相对准确、灵敏度高、特异性强、操作相对简便的方法;该方法可用于乙型肝炎患者病情监测,有效指导临床用药;联合该法与血清标志物检测,可更准确评价HBV感染者病情。     

Establishment of real-time polymerase chain reaction method for quantitative analysis of asparagine synthetase expression

We established a real-time quantitative PCR (RQ-PCR) with which to measure abundance of the asparagine synthetase (AS) mRNA. The level of AS mRNA paralleled AS enzyme activity, as well as the AS protein level detected by Western blotting and by in situ immunostaining. Cytotoxicity tests in vitro showed that the AS mRNA level also synchronized with cellular resistance to L-asparaginase in cell lines. Cellular levels of AS enzyme activity correlated with resistance to L-asparaginase. These results indicate that the AS mRNA level is an index of resistance to L-asparaginase. RQ-PCR is superior to enzyme assays, Western blotting, and immunostaining in the following ways: less labor and time, accurate and reproducible quantitativity, and broad dynamic range. In addition, RQ-PCR could evaluate differences in L-asparaginase sensitivity although immunostaining could not. And in clinical samples, we analyzed eight pediatric leukemia cases by this RQ-PCR to evaluate whether this method was applicable to clinical laboratories and the expression level of AS mRNA in each case were predictable for the effectiveness of L-asparaginase treatment. Consequently, this method was useful enough in defining candidates for selective therapy that targets an AS deficiency.     

Real-time polymerase chain reaction detection of parvovirus B19 DNA in blood donations using a commercial and an in-house assay

BACKGROUND: European regulations require testing of manufacturing plasma for parvovirus B19 (B19) DNA to limit the load of this virus to a maximum acceptable level of 10 IU/µL. To meet this requirement, most manufacturers introduced a test algorithm to identify and eliminate high‐load donations before making large manufacturing pools of plasma units. Sanquin screens all donations using a commercial assay from Roche and an in‐house assay. STUDY DESIGN AND METHODS: Between 2006 and 2009, 6.2 million donations were screened using two different polymerase chain reaction (PCR) assays targeting B19 DNA. Donations with B19 DNA loads of greater than 1 × 10⁶ IU/mL showing significant differences in viral load between the two assays were further analyzed by sequencing analysis. RESULTS: A total of 396 donations with B19 DNA loads of greater than 1 × 10⁶ IU/mL were identified. Fifteen samples (3.8%) had discordant test results; 10 samples (2.5%) were underquantified by the Roche assay, two samples (0.5%) were underquantified by the in‐house assay, and three samples (0.8%) were not detected by the Roche assay. Sequencing analysis revealed mismatches in primer and probe‐binding regions. Phylogenetic analysis showed that 12 samples were B19 Genotype 1. The three samples not detected by the Roche assay were B19 Genotype 2. CONCLUSION: This study shows that 3.8% of the viremic B19 DNA–positive donations are not quantified correctly by the Roche or in‐house B19 DNA assays. B19 Genotype 1 isolates showing incorrect test results are more common than B19 Genotype 2 or 3 isolates. Newly designed B19 PCR assays for blood screening should preferably have multiplexed formats targeting multiple regions of the B19 genome.     

实时荧光定量聚合酶链反应技术在结核性脑膜炎中的诊断价值

目的 用涂片、实时荧光定量聚合酶链反应(RT-PCR)法检测结核分枝杆菌,探讨其临床应用价值.方法 对临床确诊的结核性脑膜炎(n =110)、可疑结核性脑膜炎(n=205)和非结核性脑膜炎(n=100)患者的脑脊液标本分别采用涂片、RT-PCR两种方法进行检测,对结果进行对比分析.结果 415例脑脊液标本中,脑脊液涂片检查阳性率为2.4％(10/415),PCR检测阳性率为12.5％(52/415);脑脊液涂片、PCR法检测110例临床确诊的结核性脑膜炎患者脑脊液标本阳性率分别为6.4％(7/110)、26.4％(29/110);检测205例临床可疑结核性脑膜炎患者脑脊液标本阳性率分别为1.5％(3/205)、11.2％(23/205).比较两种方法的阳性检测率,差异有统计学意义(P＜0.05),检测100例非结核患者脑脊液标本,涂片及PCR检测均为阴性.结论 RT-PCR法检测脑脊液中TB-DNA对结核性脑膜炎的诊断有决定意义,具有较高临床应用价值.     

新型二聚体蝎型探针技术在结核分枝杆菌荧光定量聚合酶链反应中的应用

目的利用二聚体蝎型荧光探针技术，建立一种敏感特异、快速价廉且能广泛应用的结核分枝杆菌DNA荧光定量聚合酶链反应（PCR）检测方法。方法构建重组质粒pMD18-T-senX3-regX3 IR作为标准品，自行设计二聚体蝎型探针，优化定量PCR体系，并进行方法学评价及初步临床应用。结果（1）成功构建了重组质粒pMD18-T-senX3-regX3IR。（2）建立了利用二聚体蝎型荧光探针的荧光定量PCR方法，其线性范围：10^1～10^7拷贝／μ1；灵敏度：10^1拷贝／μ1；重复性：批内变异系数（CV）为2．35％，批间CV为3．17％，日间CV为4．06％；特异性100％；（3）初步临床应用证明：该方法比商品化Taqman方法阳性检出率更高，检测时间更短和成本也明显降低。结论本研究首次建立了以二聚体蝎型荧光探针技术为平台的结核分枝杆菌荧光定量聚合酶链反应（FQ-PCR）方法，这为应用二聚体蝎型荧光探针技术开辟检测结核分枝杆菌的分子诊断新途径奠定了一定的基础。     

Quantitative direct probe method for the detection of parvovirus B19

Parvovirus B19 infection is associated with anemia and spontaneous abortions. While many qualitative assays are available, a few molecular-based quantitative methods have been described. This study reports the development and optimization of a quantitative direct-probe method for the detection of Parvovirus B19 DNA. Different concentrations of RNA probes were used to identify the optimal conditions for hybridizing to the target DNA. Detection of DNA was linear between concentrations of 2 ng/ml to 200 pg/ml. Because this method requires no enzymatic amplification, it is not susceptible to amplifier contamination or enzymatic inhibitors, and it can be applied to serum samples or paraffin-embedded tissue.     

实时荧光定量PCR检测DHX32 mRNA方法的建立及初步应用

目的建立实时荧光定量PCR(FQ-RT-PCR)检测DHX32 mRNA的方法并研究其在结直肠癌中的表达。方法用Taq-Man探针建立检测DHX32 mRNA的FQ-RT-PCR方法,评价其特异性、重复性及扩增效率,并用该法检测DHX32 mRNA在结直肠癌中的表达水平。结果批内变异系数1.1%～1.9%,批间变异系数1.2%～2.5%;扩增效率为0.85,相关系数为0.9990。癌组织DHX32 mRNA表达水平中位数为1.90×10-3(四分位间距5.47×10-3),明显高于癌旁组织0.09×10-3(四分位间距1.41×10-3),两者差异有统计学意义(P<0.05)。DHX32 mRNA表达水平与结直肠癌癌栓形成、淋巴结转移、组织学分级以及Dukes分期关系密切(P<0.05)。肿瘤的恶性程度越高,DHX32 mRNA表达水平越高。结论FQ-RT-PCR检测DHX32 mRNA的方法特异性好,重复性高,操作简单,无污染。DHX32 mRNA表达上调可能在结直肠癌发生、发展中起重要作用,其表达水平可作为结直肠癌恶性程度的评价指标。