检测贝氏柯克斯体的实时荧光定量PCR

目的采用新型TaqMan-MGB探针建立检测贝氏柯克斯体的实时荧光定量PCR(real-timequantitativePCR)方法。方法根据贝氏柯克斯体特异的23SrRNA插入基因序列设计引物和探针,以克隆的23SrRNA插入基因片段作DNA模板,在荧光定量PCR检测仪(ABI7900型)上建立实时荧光定量检测方法。结果建立的定量标准曲线的循环阈值(Ct)与模板拷贝数呈良好的线性关系(r=0.997);与套式PCR相比较,荧光定量PCR检测敏感性是其100倍。用荧光定量PCR检测其它相关立克次体,检出结果均为0。用荧光定量PCR检测贝氏柯克斯体感染的小鼠脾脏标本,脾脏中贝氏柯克斯体的感染量与感染过程具有相关性。结论本研究建立的检测贝氏柯克斯体的实时荧光定量PCR具有很高的特异性和敏感性,特别适合样本中微量贝氏柯克斯体DNA的检测,其定量检测可用于动物实验中的贝氏柯克斯体感染程度的分析。     

实时荧光定量PCR检测恙虫病东方体

目的建立检测恙虫病东方体的实时荧光定量PCR(quantitative real-ti me PCR)方法。方法根据恙虫病东方体56kD外膜蛋白基因序列设计引物和探针,以克隆的56kD基因片段作DNA模板,建立实时荧光定量PCR检测方法。结果建立的荧光定量PCR标准曲线的循环阈值(Ct)与模板拷贝数呈良好的线性关系(r=0.999)。荧光定量PCR检测恙虫病东方体的灵敏度约为套式PCR的100倍,并且具有良好的重复性。用该定量PCR检测其它相关立克次体和病原菌DNA样本,检出结果均为0。用该定量PCR检测恙虫病东方体实验感染小鼠的血、脾脏、肺脏、肝脏标本,结果脾脏中东方体出现最早和检出量最多,肝脏和肺脏次之。血中的恙虫病东方体量较低。结论本研究建立的荧光定量PCR方法具有很高的特异性和敏感性,可用于东方体感染早期血样本的快速检测作恙虫病感染早期诊断,并且可以定量分析评价恙虫病东方体感染的程度。     

实时荧光定量PCR技术检测肺炎衣原体的诊断价值

目的探讨实时荧光定量PCR技术检测肺炎衣原体的诊断价值。方法对呼吸道感染的186例患者的痰液分别采用实时荧光定量PCR技术和基因测序检测,以评价实时荧光定量PCR技术检测肺炎衣原体感染的准确性。结果实时荧光定量PCR检测肺炎衣原体的灵敏度为100.0%,特异度为93.1%,阳性预期值为80.8%,阴性预期值为100.0%,准确率为94.6%。结论实时荧光定量PCR技术检测肺炎衣原体具有较高的可靠性,适用于临床快速诊断肺炎衣原体感染的相关疾病。     

实时荧光定量PCR在登革热病毒快速检测中的应用

目的应用实时荧光定量PCR快速检测登革热病毒感染。方法采集疑似登革热患者血清,采用实时荧光定量PCR检测登革热病毒,同时采用ELISA检测血清登革热IgM抗体。结果患者发病第7d血清登革热IgM血清抗体A值为0.236和0.237(临界值0.250),高度疑似阳性,第13d血清IgM抗体阳性。患者发病第2d,通用型核酸检测显示血清登革热病毒核酸含量较多,经过治疗,于第7d病毒拷贝下降。发病第2d,核酸检测确定感染病毒为登革热Ⅲ型;发病第13d血清登革Ⅲ型病毒核酸检测阴性。结论实时荧光定量PCR法具有准确性好、灵敏度高等优点,可用于登革热病毒感染的快速检测。     

实时荧光定量PCR检测肝癌患者外周血甲胎蛋白mRNA水平

在生理状态下 ,甲胎蛋白 (AFP)基因主要由胎肝细胞表达产生 ,出生后该基因表达迅速受到抑制。肝细胞大量坏死或发生原发性肝癌时 ,AFP基因再次激活[1] 。我们采用以Taqman技术为基础的实时荧光定量PCR技术 ,检测各型肝癌患者外周血细胞中AFPmRNA水平 ,分析外周血中是否存在肝癌细胞 ,并判断该方法检测肝癌细胞血液转移的可行性。一、材料与方法1 病人与标本 :本院 2 0 0 2年 8～ 12月收治的 5 7例肝癌患者 ,血清学AFP检测阳性 ,经影像学、组织病理学检查确诊为原发性肝癌。抽取肝癌住院患者晨血 5ml,EDTA抗凝。对照组为健康体检者…     

液滴式数字PCR与实时荧光定量PCR检测利什曼原虫的方法比较

目的 建立内脏利什曼病液滴式数字PCR检测方法,将其与同时建立的实时荧光定量PCR检测方法进行方法学比较,探究其在内脏利什曼病诊断方面的价值。方法 以杜氏利什曼原虫小环动基体DNA(kinetoplast DNA,kDNA)为分子靶标,根据其约200 bp的保守片段设计引物和探针,建立内脏利什曼病液滴式数字PCR检测方法,比较其与实时荧光定量PCR检测方法的检测限、精密度以及在不同利什曼原虫虫株中的检测效果。结果 液滴式数字PCR检测下限与实时荧光定量PCR检测下限相同,约为1.0×10 copies/μL;随待测样本浓度的降低(1.0×10⁴ copies/μL～1.0×10 copies/μL),液滴式数字PCR检测方法变异系数呈增大趋势(12.07%～62.96%),重复性越差;实时荧光定量PCR变异系数较小(2.96%～4.26%),且不同浓度之间的变异系数差别不大。2种方法在不同利什曼原虫中的检测灵敏度不同。结论 和实时荧光定量PCR相比,液滴式数字PCR在利什曼原虫检测中的灵敏度和重复性没有表现出显著的优势,后续还需更多的研究从分子靶标的选择、灵敏度、...     

实时荧光定量PCR技术检测食管鳞癌c-myc mRNA的表达

目的 探讨c-myc mRNA表达与食管鳞癌发病的关系.方法 采用实时荧光定量PCR法检测24例食管鳞癌患者食管鳞癌组织和正常食管鳞状上皮组织、癌旁食管鳞状上皮组织中c-myc mRNA的表达.结果 食管鳞癌组织与癌旁食管鳞状上皮组织c-myc基因的表达量相近,二者均明显高于正常食管鳞状上皮组织(P<0.05).结论 c-myc基因在食管鳞状上皮癌变过程的早期阶段起重要作用.     

人类SUZ12基因实时荧光定量PCR检测方法的建立

目的:建立SYBR Green I实时荧光PCR定量检测人类SUZ12基因的方法.方法:将SUZ12 RT-PCR扩增片段克隆入载体pGEM-T后,经测序鉴定正确后,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SUZ12,建立标准曲线,熔解曲线分析产物的特异性.结果:该法检测的最低拷贝数为14.2,线性范围为1.42×10~1-1.42×10~8拷贝,相关系数r为-1.00,1.42×10~7拷贝/L标本的批内变异系数(CV)和日间CV分别为1.8%和2.8%.熔解曲线分析显示单一的峰,熔解温度(melting temperature,Tm)为(81.37±0.16)℃.结论:实时荧光定量PCR方法检测SUZ12基因,快速有效、灵敏度高、特异性好.     

实时荧光定量PCR检测人类EZH2基因方法的建立

目的:建立SYBR Green Ⅰ实时荧光PCR定量检测人类EZH2基因的方法.方法:将EZH2 RT-PCR扩增片段克隆入载体pGEM-T后,经测序鉴定正确后,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EZH2,建立标准曲线,熔解曲线分析产物的特异性.结果:该法检测的最低拷贝数为10,线性范围为101～108拷贝,相关系数r为-1.00,104copies/μl标本的批内变异系数(CV)和日间CV分别为1.0%和2.7%.熔解曲线分析显示单一的峰,熔解温度(Tm)为(83.42±0.13)℃.结论:实时荧光定量PCR方法检测EZH2基因,具有高敏感性、高特异性和高精确性等优点,可作为进一步研究EZH2的方法.     

多重实时荧光定量PCR对下呼吸道感染病原体检测分析

目的比较多重实时荧光定量PCR(multiplex real-time polymerase chain reaction, MRT-PCR)和间接免疫荧光法(indirect immunofluorescence assay, IFA)对成人呼吸道病毒及非典型病原体检测结果。 方法收集2014年1月至2017年12月间呼吸内科210例成人呼吸道感染的标本,采用MRT-PCR检测8种常见呼吸道病原体,同时应用IFA检测血清8种病原体IgM抗体,并全部进行PCR及测序分析,比较两种方法的特异性及敏感性,评估MRT-PCR的临床应用价值。 结果210例下呼吸道感染标本经MRT-PCR和IFA检测,阳性率分别为58.57%和38.10%,混合感染率分别为7.62%和4.76%。两种方法的灵敏度分别为94.74%和35.96%,特异度分别为84.38%和59.38%。灵敏度和特异度差异有统计学意义(P<0.05)。 结论与IFA相比,MRT-PCR灵敏度、特异度好,检测性能优于IFA。     

建立16S rRNA基因聚合酶链反应方法快速检测脑膜炎奈瑟菌

目的建立以16S rRNA基因为靶基因的聚合酶链反应(PCR)方法,用于快速检测脑膜炎奈瑟菌。方法根据GenBank公布的奈瑟菌属16S rRNA基因序列,使用DNAstar软件设计引物,应用PCR方法特异检测脑膜炎奈瑟菌,对部分菌株结合16S rRNA基因测序和APIRNH生化鉴定系统以验证该方法的准确性,同时与文献报道的检测脑膜炎奈瑟菌crgA-PCR方法进行比较。结果 16S rRNA-PCR与crgA-PCR方法检测脑膜炎奈瑟菌阳性预测值皆为100%,阴性预测值分别为100%和46.94%,假阳性率分别为0%和36.73%,假阴性率皆为0%。约登指数分别为1和0.47。对188名健康儿童咽拭子杂菌DNA应用16S rRNA-PCR和crgA-PCR方法检测脑膜炎奈瑟菌,检测阳性率分别为18.62%(35/188)和15.96%(30/188)。两者同时检测到脑膜炎奈瑟菌的标本为7.98%(15/188)。结论 16SrRNA-PCR方法特异、敏感、快速,可用于脑膜炎奈瑟菌的快速检测与鉴定。     

Optimal sampling time after preparation of platelet concentrates for detection of bacterial contamination by quantitative real-time polymerase chain reaction

BACKGROUND AND OBJECTIVES: A universal quantitative real-time polymerase chain reaction (PCR), based on bacterial 16S rDNA, to detect bacterial contamination of platelet concentrates (PCs), was developed previously and compared with automated culturing. In the present study, this real-time PCR method was evaluated to determine the optimal sampling time for screening of bacterial contamination in PCs. MATERIALS AND METHODS: Routinely prepared PCs were spiked with suspensions of Escherichia coli, Bacillus cereus, Staphylococcus epidermidis, Pseudomonas aeruginosa and Propionibacterium acnes to 1, 10 and 100 colony-forming units (CFU)/ml and stored at room temperature for 7 days. The presence of bacteria in these PCs was monitored by quantitative real-time PCR. As a reference method (additional control), BacT/Alert automated culturing was used. For PCR, 1-ml aliquots were drawn from all (spiked) PCs on days 0, 1, 2, 3, 6 and 7 of storage. As a control, triplicate samples (10 ml) were inoculated into aerobic and anaerobic BacT/Alert culture bottles immediately after spiking (day 0) and after storage for 1, 2, 3, 6 or 7 days. RESULTS: With quantitative real-time PCR, all bacterial species tested were reproducibly detected on day 1 after spiking at original concentrations of 10 and 100 CFU/ml. Bacteria were also detected on day 1 from PCs spiked with an initial concentration of 1 CFU/ml, except for E. coli, which was detected in only one of the three samples and P. aeruginosa, for which analysis was not performed on day 1. With the reference method, bacteria were detected in culture bottles (inoculated on day 0) within a mean time of 20.1 h, with the exception of P. acnes which was detected at a mean time of 102.3 and 49.3 h (for original spiking concentrations of 10 and 100 CFU/ml respectively). CONCLUSIONS: PCR enables the rapid detection of low initial numbers of bacteria in PCs. For reliable detection, our results support that sampling of PCs for real-time PCR screening should not be carried out earlier than 1 day after preparation (48 h after blood collection). Importantly, the real-time PCR approach has the potential to be used before the release of PCs from the blood centre or shortly before they are transfused in the hospital.     

基于DPO引物特异性检测小肠结肠炎耶尔森氏菌的PCR方法

目的 引入一种设计简易、特异性强、退火温度范围宽的双启动寡核苷酸引物（dual-priming oligonucleotide,DPO）设计,建立基于DPO引物特异性检测小肠结肠炎耶尔森氏菌的PCR方法.方法 以小肠结肠炎耶尔森氏菌16S 23SrRNA基因为靶基因,设计一对DPO引物,经过PCR反应体系优化,建立小肠结肠炎耶尔森氏菌DPO-PCR检测方法.测定了检测灵敏度,以常规PCR方法作为参照,分析DP＆PCR方法的特异性及退火温度.结果 建立的小肠结肠炎耶尔森氏菌DPO-PCR检测方法的灵敏度为1.43×102 CFU/mL;与常规PCR方法相比,DP＆PCR方法在49～69℃退火温度范围内均能保持高效率扩增;特异性强,所测试17种病原菌中,仅小肠结肠炎耶尔森氏菌为阳性结果,且无非特异性扩增.结论 DPO-PCR方法不需要对引物参数特别是退火温度进行优化,特异性强,为致病微生物的快速准确检测提供了新方法.     

Analysis of haematopoietic chimaerism by quantitative real-time polymerase chain reaction

Allogeneic bone marrow transplantation (BMT) with marrow ablative conditioning is the treatment of choice for haematopoietic malignancies. The use of nonmyeloablative stem cell transplants has allowed the treatment of patients previously ineligible for BMT because of age or other disease. These reduced conditioning regimes allow the persistence initially of some recipient cells in the blood and bone marrow (haematopoietic chimaerism). Monitoring of the relative proportion of donor and recipient cells is required to assess the success of the procedure, to predict subsequent rejection or impending relapse and to guide the use of donor lymphocyte infusions. We present a quantitative real-time PCR approach for the measurement of haematopoietic chimaerism using the TaqMan. This approach exploits the presence of single-nucleotide polymorphisms (SNPs) to distinguish cells of patient or donor origin. We have designed and validated a panel of seven allele-specific probes to quantify the contribution of patient and donor cells in the haematopoietic population from 12 patient and donor pairs. We have compared the performance of this approach with an existing method and proved it to be superior in both accuracy and sensitivity. The use of more sensitive and accurate techniques permits earlier intervention for improved clinical outcome.     

常见分枝杆菌不同株16S-23S rRNA转录间隔区序列分析

目的 分析常见分枝杆菌不同株之间的因型,为临床分离株的鉴定提供参考序列.方法 用16S-23S rRNA转录间隔区(internal transcribed spacer,ITS)序列分析法对94株由德国微生物和细胞保藏中心(DSMZ)引进的常见分枝杆菌进行种内不同株的基因型分析,并分别与12株国际标准株对比.通过种内不同株间同源性序列比对,绘制16S-23S rRNA ITS序列聚类分析树状谱,使用DNAStar的MegAlign软件计算株间相似性百分比.结果 成功完成上述共106株菌株的16S-23SrRNA ITS测序,发现除胞内分枝杆菌(4株)、鸟分枝杆菌(4株)、海分枝杆菌(6株)和马尔摩分枝杆菌(2株)各有一个基因型且与标准株相同外,其他分枝杆菌均可分为数个不同基因型.结论 16S-23S rRNA ITS序列分析可以根据种内各株的不同基因型将分枝杆菌鉴定到株,是分枝杆菌基因型分析的可靠方法.     

Detection of apoptosis-associated microRNA in human apheresis platelets during storage by quantitative real-time polymerase chain reaction analysis

Background

Platelet transfusion is an essential part of the treatment of a variety of conditions such as thrombocytopenia and qualitative platelet disorders. As indicated in previous reports, during in vitro storage, platelets undergo morphological and physiological changes collectively known as the platelet storage lesion. Apoptosis is a programmed process of cell death, which has been considered as an important cause of platelet storage lesion under the common storage conditions in standard blood banks. Platelets are anucleate blood cells, but contain significant amounts of microRNA (miRNA, miR), which may play an important role in the regulation of gene expression. Drawing on previously published reports on cell apoptosis, we selected 49 miRNA for analysis to explore whether miRNA are of importance during the storage of platelets.

Materials and methods

We used quantitative real-time polymerase chain reaction analysis to determine the levels of expression of miRNA in apheresis platelets at different times of storage. Bioinformatics analysis was applied to explore target genes and the main functions of the selected miRNA.

Results

Our observations suggest that apheresis platelets contain large amounts of apoptosis-associated miRNA. The levels of expression of 25 miRNA remained high and ten of these miRNA showed different expression from that at day 0. Of these ten miRNA, hsa-miR-326, hsa-miR-96, hsa-miR-16, hsa-miR-155 and hsa-miR-150 were up-regulated, while hsa-miR-7, hsa-miR-145, hsa-miR-24, hsa-miR-25 and hsa-miR-15a were down-regulated. The markedly increased expression of hsa-miR-326 in all platelets is noteworthy (p<0.001).

Discussion

Since Bcl-xl and Bak1, members of the Bcl-2 family, are the targets of hsa-miR-326, our findings suggest that hsa-miR-326 may be involved in platelet apoptosis during storage.     

快速检测细菌并革兰分型的半巢式聚合酶链反应及其初步应用

目的以半巢式聚合酶链反应(PCR)检测细菌及作革兰阴、阳性分型,并与细菌培养法比较。方法以细菌16SrRNA基因为靶序列,采用一对通用引物(pm1,pm3)和一条革兰阴性型特异性引物(pm2),以半巢式PCR方法扩增实验室保留菌株的DNA并作出革兰染色分型;以人类外周血白细胞基因组DNA、HBVDNA阳性血清以及白假丝酵母菌为对照,检测此方法的特异性;采用倍比稀释菌液作敏感性实验;与细菌培养法比较,验证此方法检测临床标本的敏感性。结果对17个实验室保留菌株进行检测,以通用引物对作第1次PCR均得到371bp长度的DNA片段;再以革兰阴性菌特异引物对(pm2,pm3)作第2次PCR,9种阴性菌均得到353bp的DNA片段,而8种阳性菌未被扩增。特异性实验表明,此通用引物与人类基因组DNA、真菌及病毒无交叉反应。敏感性实验表明,采用半巢式PCR可检测出3个CFU的细菌。对120份临床标本检测,半巢式PCR检测阳性率(29.2%)显著高于细菌培养法检测阳性率(17.5%)。结论此半巢式PCR检测细菌方法,具有特异、敏感、快速的特点,并能对细菌进行革兰阴性、阳性分型,可用于临床感染性疾病的初步诊断。