Detection and amplification of Helicobacter pylori urease gene A in gastric biopsies by using nested polymerase (?)hain reaction

A nested polymerase chain reaction(N-PCR)for the spegific detection of Helicobacterpylori(H.pylori)was developed with two primer pairs(nested primers)derived from ureasegene A of H.pylori.The N-PCR was used to detect 21 different samples of H.pylori including20 clinical isolates and 1 reference strain NCTC 14126,but it was negative for other bacterialspecies,showing the N-PCR assay to be 100% specific.Tenfold serial dilution experiments re-vealed the detection of as little as 0.1 fg of H.pylori DNA by N-PCR.To evaluate the PCR as-say for clinical samples,gastric biopsies were tested with N-PCR,and the results were comparedwith those of culture,urease test and histologic examination(reference standard,RS).In 30biopsy specimens,H.pylori DNA sequences were detected by PCR in all of 20(100%)positivetissue and none of the 10 negative tissues.PCR is a specific and sensitive method that can detectthe presence of H.pylori without the need for culture and would have significant importance di-agnostically and epidemiologically.     

Construction of Eukaryotic Expression Plasmid of bFGF Gene in Rats and Its Expression in Tenocytes

The bFGF plays an important role in embryonic development of tendons and ligaments and in the healing of injuried tendons and ligaments. The eukaryotic expression plasmid of rat basic fibroblast growth factor (bFGF) gene was constructed in order to further investigate the bFGF func-tion in molecular regulatory mechanism in the repair of tendons and ligaments and to provide the foundation for the clinical application. The cDNA fragments of bFGF were cloned from the skin of rats by RT-PCR, and recombinated to the pMD18-T vector. The cDNA encoding bFGF was cloned from the pMD18-T vector by RT-PCR, digested with restriction enzyme EcoRⅠ, PstⅠ and bound to eukaryotic expression plasmid pIRES2-EGFP to construct eukaryotic expression plasmid pIRES2-EGFP-bFGF. The pIRES2-EGFP-bFGF was transfected into the tenocytes by lipid-mediated ransfection technique. MTT test was used to detect the biological activity of bFGF in supernatants after the transfection. The expression of type Ⅰ and Ⅲ collagen genes was detected by using RT-PCR. It was verified that the pIRES2-EGFP-bFGF was successfully constructed, and its transfec-tion into tenocytes could significantly enhance the biological activity of bFGF, and increase the ex-pression of type Ⅰ and Ⅲ collagen mRNA, suggesting that pIRES2-EGFP-mediated bFGF gene therapy was beneficial to the repair of tendons and ligaments.     

A study on detecting and identifying enteric pathogens with PCR

Objective To develop a rapid and definite diagnostic test of bacterial enteritis caused by pathogenic enterobacteria, the most frequent etiologic agent of infectious enteritis in the world.Methods A set of conventional PCR assays were applied to detect and identify salmonella, shigella,and E. coli O157:H7 directly from pure culture and fecal samples.The general primers of pathogenic enterobacteria were located on the uidA gene, which were found not only in E. coli nuclear acid, but also in Shigella and salmonella genes.Shigella primer was from ipaH gene whose coded invasive plasmid relative antigen existed both in plasmid and in genome.The primers of salmonella were designed from the 16SrRNA sequence.The primer of E.coli O157:H7 was taken from eaeA gene.Five random primers were selected for RAPD. The detection system included common PCR,semi-nested PCR and RAPD. Results This method was more sensitive, specific and efficient and its processing was rapid and simple. For example, the method could be used to specifically detect and identify salmonella, shigella, and E. coli O157:H7,and its sensitivity ranged from 3 to 50CFU, and its detection time was 4 hours. Conclusion This PCR method, therefore, can serve as a routine and practical protocol for detecting and identifying pathogenic microorganisms from clinical samples.     

Analysis of DNA Ploidy, Cell Cycle and Ki67 Antigen in Nasopharyngeal Carcinoma by Flow Cytometry

The expression of DNA ploidy, the cell cycle and Ki67 antigen in nasopharyngeal carcinoma (NPC) were studied and their relationship with the clinical biological behaviors and prognosis of NPC was evaluated. Biopsied specimens of NPC were made into cell suspension. By using cytometric double labeling Ki67 and DNA method, the expression of DNA ploidy, the cell cycle and Ki67 antigen were analyzed. The patients were followed up for about 3 years and the relationship between the above mentioned parameters and the clinical biological behavior and prognosis of NPC were evaluated. Of the 62 cases of NPC, the DNA aneuploid accounted for 29.03 %. The S phase cells accounted for 0 to 54 % in the cell cycle and the positive expression of Ki67 ranged from 0 to 52%. There were 40 cases of LPI (64.5%) including 15 negative cases and 22 cases of HPI (35.5%) respectively. The DNA anneploid content was positively related to the S phase cells. The patients having a low expression of Ki67 or DNA aneuploid in tumor cells were not sensitive to chemotherapy, liable to metastasis to distant organs and had a poor prognosis, while Ki67 showed no correlation with DNA ploidy and the cell cycle. It was suggested that DNA ploidy and Ki67 could be used as an independent and objective marker to evaluate the radiosensitivity and prognosis of NPC.     

Simultaneous Detection of 13 Key Bacterial Respiratory Pathogens by Combination of Multiplex PCR and Capillary Electrophoresis

Objective Lower respiratory tract infections continue to pose a significant threat to human health. It is important to accurately and rapidly detect respiratory bacteria. To compensate for the limits of current respiratory bacteria detection methods, we developed a combination of multiplex polymerase chain reaction(PCR) and capillary electrophoresis(MPCE) assay to detect thirteen bacterial pathogens responsible for lower respiratory tract infections, including Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Mycoplasma pneumoniae, Legionella spp., Bordetella pertussis, Mycobacterium tuberculosis complex, Corynebacterium diphtheriae, and Streptococcus pyogenes. Methods Three multiplex PCR reactions were built, and the products were analyzed by capillary electrophoresis using the high-throughput DNA analyzer. The specificity of the MPCE assay was examined and the detection limit was evaluated using DNA samples from each bacterial strain and the simulative samples of each strain. This assay was further evaluated using 152 clinical specimens and compared with real-time PCR reactions. For this assay, three nested-multiplex-PCRs were used to detect these clinical specimens. Results The detection limits of the MPCE assay for the 13 pathogens were very low and ranged from 10~(-7) to 10~(-2) ng/μL. Furthermore, analysis of the 152 clinical specimens yielded a specificity ranging from 96.5%-100.0%, and a sensitivity of 100.0% for the 13 pathogens. Conclusion This study revealed that the MPCE assay is a rapid, reliable, and high-throughput method with high specificity and sensitivity. This assay has great potential in the molecular epidemiological survey of respiratory pathogens.     

Specific detection of toxigenic vibrio cholerae based on in situ PCR in combination with flow cytometry

Objective To develop an in situ PCR in combination with flow cytometry (ISPCR-FCM) for monitoring cholera toxin positive Vibrio cholerae. Methods In running this method, 4% paraformaldehyde was used to fix the Vibrio cholerae cells and 1 mg/mL lysozyme for 20 min to permeabilize the cells. Before the PCR thermal cycling, 2.5% glycerol was added into the PCR reaction mixture in order to protect the integrality of the cells. Results A length of 1037bp DNA sequence was amplified, which is specific for the cholera toxin gene (ctxAB gene). Cells subjected to ISPCR showed the presences of ctxAB gene both in epifluorescence microscopy and in flow cytometric analysis. The specificity and sensitivity of the method were investigated. The sensitivity was relatively low (105 cells/mL), while the specificity was high. Conclusion We have successfully developed a new technique for detection of toxigenic Vibrio cholerae strains. Further study is needed to enhance its sensitivities. ISPCR-FCM shows a great promise in monitoring specific bacteria and their physiological states in environmental samples.     

Detection of genetically modified crops by combination of multiplex PCR and low-density DNA microarray

Objective To develop a technique for simultaneous detection of various target genes in Roundup Ready soybean by combining multiplex PCR and low-density DNA microarray. Methods Two sets of the multiplex PCR system were used to amplify the target genes in genetically modified （GM） soybean. Seventeen capture probes （PCR products） and 17 pairs of corresponding primers were designed according to the genetic characteristics of Rroundup Ready soybean （GTS40-3-2）, maize （MonS10, Nk603, GA21）, canola （T45, MS1/RF1）, and rice （SCK） in many identified GM crops. All of the probes were categorized and identified as species-specific probes. One negative probe and one positive control probe were used to assess the efficiency of all reactions, and therefore eliminate any false positive and negative results. After multiplex PCR reaction, amplicons were adulterated with Cy5-dUTP and hybridized with DNA microarray. The array was then scanned to display the specific hybridization signals of target genes. The assay was applied to the analysis of sample of certified transgenic soybean （Roundup Ready GTS40-3-2） and canola （MS1/RF1）. Results A combination technique of multiplex PCR and DNA microarray was successfully developed to identify multi-target genes in Roundup Ready soybean and MS 1/RF1 canola with a great specificity and reliability. Reliable identification of genetic characteristics of Roundup Ready of GM soybean from genetically modified crops was achieved at 0.5% transgenic events, indicating a high sensitivity. Conclusion A combination technique of multiplex PCR and low-density DNA microarray can reliably detect and identify the genetically modified crops.     

Evaluation of Carotid Atherosclerotic Plaque Stability with Contrast-enhanced Ultrasonography

This study was aimed to evaluate the relationship between carotid atherosclerotic plaque stability and the clinical symptoms in patients with carotid atherosclerotic plaques by using contrast-enhanced ultrasonography. Fifty patients with carotid atherosclerotic plaques were enrolled and examined with contrast-enhanced ultrasonography. The correlation of contrast agent enhancement of the carotid atherosclerotic plaques and the clinical symptoms was analyzed. The results showed that among the 50 patients, plaques were enhanced in the 23 patients with obvious clinical symptoms. In 27 patients without apparent clinical symptoms, plaques were enhanced sparsely in 15 patients and not enhanced in 12 patients. It was suggested that contrast-enhanced ultrasonography could be used for the examination of the microcirculation in carotid atherosclerotic plaques on real-time basis and serve as a new noninvasive approach for the assessment of stability of carotid atherosclerotic plaques.     

Establishment and analysis of specific DNA patterns in 16S-23S rRNA gene spac er regions for differentiating different bacteria

Objective To establish the specific 16S-23S rRNA gene spacer regions in different bacteri a using polymerase chain reaction (PCR), restriction fragment length polymorphis m (RFLP), DNA cloning and sequences analysis. Methods A pair of primers were selected from highly conserved sequences adjacent to the 16S-23S rRNA spacer region. Bacterial DNA from sixty-one strains of standard bacteria and corresponding clinical isolates representative of 20 genera and 26 species was amplified by PCR, and further analyzed by RFLP, DNA cloning and se quences analysis. Furthermore, all specimens were examined by bacterial cultur ing and PCR-RFLP analysis. The evaluation of these assays in practical clinic practice was also discussed. Results Restriction enzyme analysis revealed one, two or three bands or more observed am ong the 26 different standard strains. The sensitivity of PCR reached 2.5 colony-forming unit (CFU), and there was no cross reaction with human genomic DNA, fungus or virus. Fourt ee n species could be distinguished immediately by PCR, while another 10 species we re further identified by Hinf Ⅰ or Alu Ⅰ digestion. The only difference betwe en K.pneumoniae and E.durans was located at the site of the 779th nucleot ide according to the sequence analysis and only XmaⅢ digestion could distingui sh one from another. Of 42 specimens from septicemic neonates, 15 were identifi e d as positive by blood culture at a rate of 35.7%. However, 27 specimens ident ified as positive by PCR, with a rate of 64.2%, a method significantly more eff ective than blood culture (P＜0.01). Of 6 cerebrospinal fluid (CSF) specim ens, one tested positi ve for S.epidermidis was also positive by PCR, two culture negative were po sitive by PCR and diagnosed as S.epidermidis according to the DNA pattern. One positive for C.neoformans was negative by PCR. The other two specimen s were negative by both PCR and culture. Conclusions The method of detecting bacterial 16S-23S rRNA spacer regions using PCR-RFLP t echniques was specific, sensitive, rapid and accurate in providing a new techniq ue for detecting pathogens in clinical bacterial infections.     

TaqMan实时荧光定量RT-PCR检测外周血CK19 mRNA

目的:应用TaqMan实时荧光定量RT-PCR技术,建立定量检测CK19 mRNA的方法. 方法:采用RT-PCR从胃癌细胞中克隆CK19片段(230 bp),装入pMD 18-T Simple载体.纯化质粒,制备荧光定量PCR标准品.应用LightCycle荧光定量PCR仪检测标准品、30例正常人外周血和5例肿瘤组织CK19 mRNA. 结果:PCR扩增及测序均证实CK19 cDNA片段重组到pMD 18-T载体上,建立了稳定的检测CK19 mRNA的标准,即设定CT值在35个循环之内,检测结果低于100个拷贝数量级为阴性标本.结论:采用TaqMan实时荧光定量RT-PCR技术检测外周血CK19 mRNA是一种稳定可靠的方法.     

CAR菌TaqMan MGB探针法实时荧光定量PCR快速检测方法的建立与应用

目的建立特异、敏感、快速检测CAR菌的TaqMan MGB探针荧光定量PCR方法。方法针对CAR菌16S rRNA基因序列设计特异性引物和探针,建立MGB探针荧光定量PCR方法,验证方法的特异性、敏感性和稳定性。对2008～2012年期间采集的1 344份临床标本中的CAR菌进行检测,同时进行普通PCR检测作为对照。结果 CAR菌TaqMan MGB探针荧光定量PCR方法具有高度特异性,与肺炎支原体、侵肺巴斯德菌、支气管鲍特杆菌、肺炎克雷伯杆菌、大肠埃希菌、肺炎链球菌间无交叉反应,检测灵敏度达2.7拷贝。标准曲线显示各浓度范围内具有良好线性关系,相关系数为0.999,斜率为-3.22,TaqMan MGB探针荧光定量PCR效率为104.432%。对1 344份标本进行检测,结果 TaqMan MGB探针荧光定量PCR检出510份CAR菌阳性样本。TaqMan MGB探针荧光定量PCR能够直接从标本中检出CAR菌DNA,检测时间仅为40 min。结论 TaqMan MGB探针荧光定量PCR方法具有可靠、特异、敏感的特点,适用于CAR菌的快速检测。     

TaqMan 探针荧光定量PCR支原体快速检测方法的建立及应用

目的： 在支原体16SrRNA的种属特异性区域设计一对引物及其相应的Taqman探针,建立荧光定量PCR方法,检测细胞培养物中支原体。方法：选择16srRNA支原体种属特异性区域作为扩增区,设计支原体通用引物,PCR扩增片段的预期大小为288 bp。回收PCR扩增产物,连接pMD-18T载体,转化JM109大肠杆菌,涂布Amp+琼脂平板,挑取阳性克隆进行PCR鉴定后提取质粒。构建荧光定量PCR绝对定量的标准品,设计荧光定量引物、探针,采用矩阵法对反应体系进行优化,建立绝对定量的标准曲线。利用十倍稀释法检验灵敏度并与普通PCR法及培养法进行比较;对支原体及其他5种革兰氏阳性杆菌进行特异性检验;对培养法、普通PCR法及荧光定量PCR法检测样本的灵敏度进行比较。结果：支原体PCR产物大小约为288 bp,质粒质量浓度为13.9 mg/L,A260/Ａ280 (Ratio)为1
.780,质粒溶液浓度为4.25×109拷贝/ μL。10 μmol/L的引物浓度和10 μmol/L-1探针浓度为最佳反应浓度,双温循环及60℃退火温度为最佳的循环条件。检测灵敏度为4.250×101 拷贝。拷贝数与Ct值之间的线性关系表达式：Ct= －2.916×log拷贝数+40.02。特异性检验中5种革兰氏阳性杆菌样本检测均为阴性,支原体样本为阳性,表明特异性好。准确性检测中变异系数小,表明稳定性好。组内及组间相同浓度样本检测具有相同的Ct值,表明重现性好。样本检验中,荧光定量PCR检测出的阳性样本高于普通PCR及培养法。结论：支原体荧光定量PCR法检测细胞样本及临床样本中支原体快速、特异性强、灵敏度高、稳定性好。     

实时荧光定量TaqMan-PCR检测小鼠多瘤病毒方法的建立

目的 建立一种快速、特异、敏感的小鼠多瘤病毒 (murine polyomavirus, MPyV)荧光定量 TaqMan PCR检测方法, 用于MPyV核酸的检测。 方法 根据GenBank中小鼠多瘤病毒基因组序列设计了一对特异性引物及TaqMan探针, 扩增长度为69 bp的片段, 通过优化反应体系和反应条件, 对构建的重组质粒标准品进行检测, 并绘制标准曲线, 进行特异性、敏感性和重复性试验。最后用建立的方法对人工感染MPyV的肺脏、脾脏和粪便, 及86 份小鼠临床样本进行检测, 验证在临床应用中的效果。 结果 所建立的检测方法特异性强, 只能在小鼠多瘤病毒DNA中检出荧光信号, 检测灵敏度达到100拷贝, 批内和批间重复性好, 检测结果变异系数(CV)均小于1.13%, 人工感染MPyV小鼠的肺脏、脾脏和粪便均为阳性, 对86份临床样本进行检测, 有3份样本呈阳性(阳性率为3.5%)。 结论 建立的MPyV荧光定量TaqMan-PCR检测方法特异性强、敏感性高、重复性好, 适合用于MPyV临床诊断、日常监测和流行病学调查。     

鸭疱疹病毒1型实时荧光定量PCR方法的建立

目的建立检测鸭疱疹病毒1型(鸭瘟病毒)的实时荧光定量PCR方法,为快速诊断、致病机理研究及抗病毒药物筛选等奠定基础。方法根据病毒DNA聚合酶基因的序列,设计引物和探针,采用TaqMan探针技术进行实时荧光定量PCR,用含有125bp扩增产物的pMD18-T载体质粒为阳性对照,构建标准曲线,对该方法的特异性、可重复性、敏感性进行评价,同时与传统PCR方法进行比较研究。结果标准曲线表明在2.3×105~2.3×10拷贝数之间有很好的线性关系(r=0.999);实时荧光定量PCR最少可检测到23个阳性质粒,说明有很好的敏感性;试验内及试验间变异系数分别为1.22~6.69以及2.09~8.84,说明有较好的重复性;对非鸭瘟病毒DNA无扩增,说明有很好的特异性;在对病毒DNA的检测方面,比传统PCR的敏感性高出104倍。结论成功建立了针对鸭瘟病毒的特异、敏感、重复性强且可准确定量的实时荧光定量PCR方法。     

胆汁螺杆菌实时荧光定量PCR检测方法的建立

目的 建立快速、敏感、特异的胆汁螺杆菌(Helicobacter bilis,H.bilis)TaqMan探针实时荧光定量PCR检测方法,对胆汁螺杆菌进行定量检测。方法 PCR扩增H.bilis保守基因P17序列全长ORF435 bp,进行TA克隆,构建质粒标准品pMD-HBP17。通过对pMD-HBP17标准品的定量分析,优化反应体系,检测TaqMan探针实时荧光定量PCR方法的灵敏度、特异性及重复性;用所建立的qPCR方法检测77份临床样品,并与普通PCR的检测结果作比对。结果 所建立的qPCR检测方法,质粒DNA浓度在10⁸~10¹拷贝之间表现出较好的线性和相关性,所得标准曲线的斜率为-3.46,相关系数为0.999,检测灵敏度达到20个拷贝,对77份临床样品的检出率为14.3%,较普通PCR(7.8%)的检出率高。结论 建立的H.bilis qPCR检测方法特异性好、敏感性高、稳定性强,可用于胆汁螺杆菌的定量及定性检测。     

博尔纳病病毒荧光定量套式RT-PCR检测方法的建立

目的：建立博尔纳病病毒(BDV)荧光定量套式RT-PCR检测方法，为快速、准确地定量检测BDV奠定基础。方法：根据BDV P24基因序列，设计并合成引物和荧光标记探针。将PCR扩增的BDV P24基因片段克隆入载体，重组质粒经筛选、鉴定后，作为阳性模板，用于标准曲线的制定和样品检测。结果：应用量组质粒制作的定量曲线循环阈值与模板具有良好的线性关系，相关系数为0．998，建立了检测BDV的荧光定量套式RT-PCR方法。检测58例神经精神病患者及50例健康人外周血单核细胞(PBMC)中BDV P24基因片段，3例精神分裂症及1例帕金森病患者呈阳性，其余均为阴性。结论：荧光定量套式RT-PCR方法能够避免PCR后处理导致的假阳性污染，并实现准确定量，对于研究BDV感染与人类神经精神疾病的关系有很好的应用价值。     

双重实时荧光PCR检测副溶血性弧菌和创伤弧菌方法的建立

目的建立双重实时荧光PCR检测副溶血性弧菌和创伤弧菌的方法。方法在Gen Bank数据库查找序列并比对后设计tdh和vvh A基因引物和探针。对PCR反应退火温度、引物、探针、Mg2+、Taq DNA聚合酶及d NTPs浓度进行优化,确定反应体系和反应条件,并对新建方法的特异性、灵敏度等方面进行分析,对模拟样品进行检测。结果建立了双重实时荧光PCR检测副溶血性弧菌和创伤弧菌的方法。该方法的特异性较好,检测限为103 CFU/ml。结论建立的双重荧光PCR方法可快速、灵敏、特异地检测副溶血性弧菌和创伤弧菌。     

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。     

Real-time PCR与常规PCR在Q热立克体检测中的比较

目的比较与评价Real-time PCR与常规PCR,寻求适合于Q热患者早期诊断的方法。方法应用Taq Man探针定量PCR、SYBR Green染料定量PCR、巢式PCR和普通PCR四种方法分别对T-A克隆的Q热立克次体基因htp AB片断进行灵敏性、特异性和重复性检测及评价。结果 Taq Man PCR方法的灵敏度分别为SYBR Green PCR、巢式PCR和普通PCR的10倍、10倍和100倍。重复性测试Taq Man法Ct变异系数CV:0.2%~3.0%,SYBR Green法的CV:0.3%~6.0%。Taq Man和SYBR Green PCR均耗时约1h,巢式PCR耗时约2.5h,普通PCR耗时约1.5h。四种方法检测25种相关病原菌结果均为阴性。结论 Taq Man探针定量PCR方法具有很好的特异性、灵敏性、重复性和可操作性,可用于Q热患者临床早期诊断。