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Objective To establish an assay for the detection of Streptococcus pneumoniae by real-time fluorescence quantititive polymerase chain reaction (PCR).Methods Special primers and probe for the autolysin A (lytA)gene were designed.The sensitivity and specificity of primers and probe were studied,and cut-off of cycle threshold was assayed.158 clinical specimens were confirmed by real-time fluorescence quantitative PCR and bacterial culture method.Results Primer and probe design for LytA gene could sensitively detect serotype Streptococcus pneumoniae strains of common pathogenic,and the sensitivity was 100 copies.Among 35 strains of Streptococcus pneumoniae,34 cases were detected to be positive for Streptococcus pneumoniae by real-time fluorescence quantitative PCR,while 1 case was detected to be negative;among 15 strains of non-Streptococcus pneumoniae, all were detected to be negative.Among the 158 clinical sputum specimens,34 cases with Streptococcus pneumoniae were detected by real-time fluorescence quantitative PCR,while only 10 cases with Streptococcus pneumoniae were detected by the culture method.White blood cells count and time in hospital of cases with Streptococcus pneumoniae were higher than those of cases without Streptococcus pneumoniae (P <0.05 ). Conclusion Real-time fluorescence quantitative PCR is a sensitive and specific assay for the detection of Streptococcus pneumoniae.It can be used for the diagnosis of Streptococcus pneumoniae.
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Objective:This study aims to select the more suitable testing method for early screening of uterine cervical cancer to protect susceptible populations. Application value was compared between the two methods of high-risk HPV detection in early screening of uterine cervical cancer. Methods:The two methods, namely, fluorescence quantitation polymerase chain reaction(PCR) and HC2-HPV-DNA, were used to detect the infection status of 13 high-risk HPV types during women's health examination. The examined women were divided into four groups according to age (23 to 29 years old, 30 to 39 years old, 40 to 49 years old, and 50 to 58 years old). Statistical methods were applied to analyze the results. Results:The detected positive rates by fluorescence quantitation PCR and HC2-HPV-DNA were 15.93%(140/879) and 11.83%(104/879), respectively, among the 879 examined women. The common positive and negative rates were 9.56%(84/879) and 81.80%(719/879), respectively. The results of the two methods showed that the infection positive rate was obviously higher in the 40 to 49 year old and 50 to 58 year old groups. Statistical difference was observed between fluorescence quantitation PCR and HC2-HPV-DNA in detecting high-risk HPV types (P40 years old who are at high risk of HPV, to prevent uterine cervical cancer efficiently.
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Objective To establish a rapid, sensitive, and specific quantitative method to detect hepatitis C virus. Methods A primer set targeting HCV 5'UTR was designed. The isothermal amplification was performed by the Bst DNA polymerase and AMV reverse transcriptase, under the temperature of 60℃ for 60 min. The signal was monitored by SYBR Green Ⅰ. Results One hundred and twenty positive serum samples, confirmed by the real-time PCR. All were detected by the isothermal amplification, while 110 healthy subjects' samples were negative by the both methods. The lower detect limit was determined to 10 IU/ml HCV-RNA, by the assay on serial dilutions of the quality control standards obtained from clinical investigation center of MOH. Conclusion A real time reverse loop-mediated isothermal amplification method was developed to detect HCV, with the characteristic of rapidity, high sensitivity and specificity.
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Objective To study the adiponectin mRNA expression in subcutaneous and omental adipose tissues of type 2 diabetes and their relation with blood adiponectin, body weight index, waist hip ratio and insulin sensitivity. Methods The adiponectin mRNA expression level in adipose tissue of 22 cases with type 2 diabetes and 26 cases of non diabetes controls was assayed by real time fluorescence quantitative RT PCR. The level of plasma adiponectin was measured with ELISA kit. Results The adiponectin mRNA expression in omental adipose tissue was significantly decreased in patients with type 2 diabetes than that in non diabetes controls (P