核酸扩增荧光检测技术(PCR)检测肺炎支原体(MP)在社区幼儿获得性肺炎辅助诊断中的意义

目的肺炎支原体(MP)核酸扩增荧光检测技术(PCR)测定在社区幼儿获得性肺炎早期辅助诊断的意义。方法收集玉林市二医院住院及社区幼儿门诊就诊患呼吸道感染及发热幼儿310例,通过三种不同方法即PCR法、血清法和培养法测定MP-DNA及MP-Ab,分析这三种方法测定结果的时间性、敏感性、特异性。评价PCR早期测定方法诊断的可行性。结果 PCR早期测定MP方法与血清法比较有统计学意义(P<0.5),与培养法比较无统计学意义(P>0.5)。结论核酸扩增荧光检测技术(PCR)可作为检测社区幼儿肺炎支原体(MP)早期检测诊断指标。

结核分枝杆菌DNA检测在肺部疾病患者临床诊断及治疗中的指导意义

目的:探讨结核分枝杆菌DNA检测在临床诊断及治疗中的指导意义。方法:选取肺部疾病患者1 000例,收集患者胸膜炎的胸水标本与咳痰标本,根据患者临床症状及涂片检测等结果将1 000例患者分为初治涂阳肺结核组(271例),初治涂阴肺结核组(265例),肺外结核组(266例)和肺部其他疾病组(198例)。另随机抽取医院80例医护人员的健康血样标本设为正常对照组。所有研究对象均采取双盲法诊断PCR扩增与ABI-7500仪器荧光检测结核分枝杆菌DNA定量。结果:271例初治涂阳肺结核组患者阳性检出率为85.98%,特异性96.25%,检测敏感性85.98%,阳性预测值98.73%,阴性预测值66.96%;265例初治涂阴肺结核组患者结核杆菌DNA阳性检出率为41.89%,特异性96.25%,检测敏感性41.89%,阳性预测值97.37%,阴性预测值33.33%;266例肺外结核组患者结核杆菌DNA阳性检出率为33.83%,特异性96.25%,检测敏感性33.83%,阳性预测值96.77%,阴性预测值30.43%。结论:采用结核分枝杆菌DNA检测具有极高的特异性与敏感度,提高了结核病患者的诊断准确率,为临床防治结核病提供重要依据。

三种检测方法在阴道分泌物真菌感染中应用分析

目的:比较常规镜检法、荧光PCR核酸扩增法(PCR法)和念珠菌显色培养基培养鉴定(真菌显色培养法)三种方法对阴道分泌物真菌检测结果的差异。方法:选取2017年1月至2017年12月就诊于本院的妇科阴道炎患者,收集常规镜检法真菌阳性和阴性标本各50例。用PCR法鉴定阳性标本念珠菌类型,阴性标本用荧光PCR核酸扩增法重新鉴定。同时将35例PCR法检测结果为阳性的标本进行真菌显色培养法培养鉴定,比较三者的结果。结果:50例常规镜检法阳性的标本,采用PCR法鉴定阳性47例,以白色念珠菌为主,占37例,光滑假丝酵母菌次之,占5例,热带假丝酵母菌2例,白色念珠菌合并光滑假丝酵母菌1例,其他真菌2例。50例常规镜检法阴性标本中,采用PCR法鉴定阳性15例,其中白色念珠菌13例,光滑假丝酵母菌1例,热带假丝酵母菌1例。常规镜检法和PCR法阳性率差异有统计学意义(P<0.05);35例样本中,有1份样本经PCR法鉴定为热带假丝酵母菌,而显色培养基培养未鉴定出,真菌显色培养法与PCR法阳性率比较无统计学差异(P>0.05)。结论:三种检测方法各有利弊,常规镜检法应为门诊常规检验中的首选。对于临床上有典型症状而常规镜检法阴性的患者,建议采用PCR法或真菌显色培养法培养检测,以提高检测准确性和阳性率。

三种检测方法对支原体检出率的对比及用药指导

目的评价三种检测方法在性病支原体检测上的优缺点及临床用药的知道意义。方法 A组应用多功能高倍显微分析系统,对泌尿生殖道患者的分泌物标本进行支原体活体动态观察、分析、定性。B组应用解脲支原体(UU)核酸扩增荧光定量检测法对泌尿生殖道患者的分泌物标本进行支原体定性、定量分析。C组应用支原体IST 2试剂盒进行UU培养基药敏分析。结果 A组能在极短时间内(约10 min)快速、准确地检测出支原体,阳性率为54.9%;B组亦能在较短时间内(约5 h)准确地检测出支原体,阳性率为61.1%,但对实验室有较高的要求,技术的误差也容易有假阴性及假阳性结果,C组此法为推荐的检测支原体的金标法,检测时间相对较长,检出率相对较低,阳性率为47.2%。但因其有药敏记过,对于临床用药有决定性的作用。结论医务工作应合理应用不同的检测方法,并根据药敏试验情况,指导临床及时、合理、规范用药,以减少耐药菌株的出现。

Novel biosensor-based microarray assay for detecting rs8099917 and rs12979860 genotypes

AIM:To evaluate a novel biosensor-based microarray(BBM) assay for detecting rs12979860 and rs8099917 genotypes.METHODS:Four probes specific for rs8099917C/T or rs12979860G/T detection and three sets of quality control probes were designed,constructed and arrayed on an optical biosensor to develop a microarray assay.Two sets of primers were used in a one tube polymerase chain reaction(PCR) system to amplify two target fragments simultaneously.The biosensor microarray contained probes that had been sequenced to confirm that they included the rs8099917C/T or rs12979860G/T alleles of interest and could serve as the specific assay standards.In addition to rehybridization of four probes of known sequence,a total of 40 clinical samples collected from hepatitis C seropositive patients were also tested.The target fragments of all 40 samples were amplified in a 50 μL PCR system.Ten μL of each amplicon was tested by BBM assay,and another 40 μL was used for sequencing.The agreement of the results obtained by the two methods was tested statistically using the kappa coefficient.The sensitivity of the BBM assay was evaluated using serial dilutions of ten clinical blood samples containing 10 3-10 4 white cells/μL.RESULTS:As shown by polyacrylamide gel electrophoresis,two target segments of the interleukin 28Bassociated polymorphisms(SNPs) were successfully amplified in the one-tube PCR system.The lengths of the two amplified fragments were consistent with the known length of the target sequences,137 and 159 bps.After hybridization of the PCR amplicons with the probes located on the BBM array,the signals of each allele of both the rs8099917 SNPs and rs12979860 SNPs were observed simultaneously and were clearly visible by the unaided eye.The signals were distinct from each other,could be interpreted visually,and accurately recorded using an ordinary digital camera.To evaluate the specificity of the assay,both the plasmids and clinical samples were applied to the microarray.First,30 PCR amplicons of the various SNP alleles were hybridized on the BBM microarray.Full agreement between plasmids and the BBM assay was observed,with 30/30 correct matches(100%).The kappa value for the BBM assay with plasmids was 1.00(P < 0.05).For the 40 clinical blood samples,the BBM assay hybridization and direct sequencing results were compared for each amplicon.For patient blood samples,agreement was 28/28 for rs8099917T/T,9/11 for rs8099917T/G,1/1 for rs8099917G/G,24/24 for rs12979860C/C,11/14 for rs12979860C/T,and 2/2 for rs12979860T/T.Only five clinical samples of amplicon assay and direct sequencing results were discordant and heterozygotes:2/11 rs8099917T/G and 3/14 rs12979860C/T.The agreement of outcomes between BBM assay and direct sequencing for the detection of rs8099917 and rs12979860 was 95% and 92.5%,respectively;and the corresponding kappa values were 0.88 and 0.85(A kappa value > 0.75 was defined as substantial agreement).The BBM assay and sequencing had similar specificities for detection and identification of the two SNPs and their alleles.The sensitivity evaluation showed that the BBM assay could detect and identify SNP sequences present in blood samples containing as few as 10 2 white blood cells/μL.CONCLUSION:This biosensor microarray assay was highly specific,sensitive,rapid and easy to perform.It is compatible with clinical practice for detection of rs8099917 and rs12979860.

Mutations of mitochondrion DNA in mouse tumors

Objective： To ascertain the variations of mitochondrion DNA （mtDNA） in mouse tumors and to inquire into the relationship between mutations of mtDNA and carcinogenesis Methods： The variations of D-loop, ND3 and tRNA^Met＋Glu＋Ile gene fragments of mtDNA from six tumor cell lines of mice were analyzed by PCR technology with restriction fragment length polymorphism analysis （polymerase chain reaction-restriction fragment length polymorphism, PCR-RFLP） and single strand conformation polymorphism analysis （SSCP-PCR） method. Results： ND3 and tRNA^Met＋Glu＋Ile gene fragments ofmtDNA from the tumors showed no variation in 27 endonuclease sites; D-loop ofmtDNA from Hca-F had an additional endonuclease sites of Hinf I in contrast to that of the inbred mouse. Deeply analyzed by PCR-SSCP, the D-loop ofmtDNA was found to possess mutations in 4 of 6 tumors. Conclusion： D-loop is the hot spot of tumor mtDNA mutation which can act as contributors to the carcinogenic

ICC-qPCR病毒灭活验证方法的建立及其应用

目的:建立细胞培养-核酸扩增荧光定量PCR(integrated cell culture quantitative PCR,ICC-qPCR)病毒灭活验证方法;结合传统细胞病变效应(cytopathic effect,CPE)法,考察ICC-qPCR方法的适用性;利用这2种方法验证复用电生理导管环氧乙烷病毒灭活工艺的有效性。方法:通过制备指示病毒(伪狂犬病毒,PRV)质粒DNA作为定量标准品,用PRV特异性引物建立定量PCR方法;再用PRV病毒侵染宿主细胞,同时用热灭活PRV作为对照,确定PRV活病毒全部进入宿主细胞但不扩增的最佳样本收获时间,通过检测细胞中活病毒,建立ICC-qPCR活病毒检测方法;确立CPE法测定滴度与ICC-qPCR法检测DNA拷贝数之间的线性关系。最后,模拟临床使用的最坏情况对一次性电生理导管进行含指示病毒(PRV)生物污染物负载,模拟环氧乙烷病毒灭活工艺,通过ICC-qPCR和CPE法验证病毒灭活工艺的有效性。结果:本研究中建立的ICC-qPCR方法的标准曲线线性关系r2>0.99;定量下限为104 copies·μL^(-1),约相当于2 logs·mL^(-1),检测下限为103copies·μL^(-1),约相当于-1 logs·mL^(-1);特异性结果表明只能检测到PRV的扩增曲线,无非特异性扩增;重复性试验的RSD<5%。确定ICC-qPCR方法中PRV最佳收获时间为宿主细胞染毒后1～2 h。ICC-qPCR检测拷贝数与病毒滴度间具有良好的线性关系(r2>0.99)。ICC-qPCR法病毒灭活工艺验证结果表明:经环氧乙烷灭活后病毒负载导管其病毒滴度降低>9.0 logs;阴性对照组(负载不含PRV生物污染物)和空白导管样品均未检出病毒DNA。CPE法的病毒滴定结果表明,经环氧乙烷灭活后病毒负载导管其病毒滴度降低≥8.5 logs·mL^(-1);阴性对照组和空白导管样品均未检出PRV。结论:本研究建立的ICC-qPCR方法应用于PRV病毒灭活验证,可以有效检测活病毒,比传统CPE法(-0.5 logs·mL^(-1))灵敏度高。将此方法应用于复用电生理导管的环氧乙烷病毒灭活工艺验证,同时与CPE法进行比较,验证了ICC-qPCR方法的适用性以及环氧乙烷灭活电生理导管负载的PRV病毒的工艺有效性。

结核分枝杆菌DNA分子生物学检测

目的探讨利用结核分枝杆菌的核酸扩增(PCR)技术,检测结核杆菌DNA在临床诊断中的价值。方法采用病例对照研究方法,各组诊断采用双盲法,对健康对照组血标本,胸膜炎的胸水标本、腹膜炎的腹水标本、脑膜炎的脑脊液标本和有泌尿系症状的尿液标本,咳痰患者的痰标本进行PCR扩增及ABI-7300仪器荧光检测结核分枝杆菌DNA定量。应用统计学分析阳性率、敏感性、特异性及各组间的统计学意义。结果共检测427例,其中初治涂阳肺结核100例,初治涂阴肺结核100例,肺外结核100例,肺部其他疾病77例,健康对照50例。结核杆菌DNA检测初治涂阳肺结核的阳性率86.0%、敏感性86.0%、特异性96.0%;阳性预测值97.7%、阴性预测值77.4%。初治涂阴肺结核的阳性率42.0%、敏感性42.0%、特异性96.0%;阳性预测值95.5%、阴性预测值45.3%。肺外结核的阳性率34.0%、敏感性34.0%、特异性96.0%。阳性预测值94.4%、阴性预测值42.1%。结论结核分枝杆菌DNA分子生物学检测技术是对传统诊断活动性结核病实验室诊断方法的补充,具有较高的敏感性和特异性,对结核病的诊断有重要的诊断价值,成为结核病诊断和研究的重要手段。