A FQ-RT-PCR method for quantitative determination of IL-2 mRNA and IL-4 mRNA and its application

The purpose of the study is to establish a fluorescence quantitative reverse transcription polymerase chain response （FQ-RT-PCR） method for the quantitative determination of IL-2 mRNA and IL-4 mRNA in Th cells, with which the Th cells status of the patients with gynaecological tumors and chronic renal failure （CRF） can be analyzed. IL-2 eDNA and IL-4 eDNA were prepared, and the plasmid pMD18 carrying IL-2 eDNA or IL-4 eDNA fragment was constructed and cloned as the template for quantitative determination. The primers and probes labelled with 6-carbexy-fluorescein （FAM） and 6-carboxy- tetramethylrhodamine （TAMRA） were prepared, and the experimental conditions were optimized to set up the FQ-RT-PCR method for quantitative determination of IL-2 mRNA and IL-4 mRNA. Th cells enriched from peripheral blood mononuclear cells （PBMCs） of 20 healthy volunteers （HVs）, 16 gynaecological benign （GB） cases, 18 gynaecological malignant （GM） tumor cases and 16 chronic renal failure （CRF） patients were tested for IL-2 mRNA and IL-4 mRNA by FQ-RT-PCR. The house-keeping gene β-actin was used as the internal control gene of the experiment. The standard curve for log concentration of series of quantitative templates vs threshold cycle （CT） was established by linear regression, and the linear range was 102-107 copies/μl. The imprecision test showed the CV of inter-assay and intra-assay of a high cont- ent sample by FQ-RT-PCR were 7.8% and 12.5%, respectively. The CV of inter-assay and intra-assay of a low content sample were 10.8% and 19.5%, respectively. The IL-2 mRNA expressions in Th of the patients with gynaecological malignant tumor （compared with the HVs and the patients with gynaecological benign disease） and in Th of the CRF patients （compared with the HVs） were declined significantly and at the same time the IL-4 mRNA expression increased significantly （ P 〈 0. 001 ）. A simple, sensitive and accurate FQ-RT-PCR method for the quantitative detection of IL-2 mRNA and IL-4 mRNA has been established. The IL-2 mRNA and IL-4 mRNA expressions in Th cells of the patients with gynaecological malignant tumor and the CRF patients were polarized and displayed Th2 response. It suggests that the function of the Th cells of the patients with gynaecological malignant tumor or CRF is at unbalance.

快速免疫层析法检测生殖道沙眼衣原体的评价及临床应用

目的 评价快速免疫层析法检测生殖道沙眼衣原体的应用价值及临床意义。方法 对681例皮肤性病科门诊和妇科门诊病人用不同的方法留取标本,同时采用快速免疫层析法和FQ -PCR法进行生殖道沙眼衣原体检测。结果 在女性第一种和第二种留取标本方法中,快速免疫层析法沙眼衣原体检出的灵敏度和特异性分别为84.2 1%、98.18%和82 .5 2 %、98.2 5 % ;男性分别为80 .82 %、97.72 %和72 .60 %、91.5 2 %。结论 快速免疫层析法检测生殖道沙眼衣原体,如果留取标本得当,具有特异、敏感、快速、简便,不需要特殊仪器设备等优点,对沙眼衣原体感染的快速诊断和治疗具有重要的临床意义。

解脲支原体检测方法比较与探讨

比较荧光定量PCR(FQ-PCR)法,培养法以及ELISA法在检测解脲支原体中诊断与方法学。对534例疑似病例取尿道或宫颈分泌物同时进行FQ-PCR法,培养法以及ELISA法对比检测。以两种方法阳性为真阳性,两种方法皆阴性为真阴性。ELISA法敏感性为81.2%,特异性为87.8%。培养法敏感性为93.4%,特异性为100%。FQ-PCR敏感性与特异性皆为100%。ELISA法特异性和敏感性都稍差。荧光PCR法检测支原体有较好的特异性和准确性,快速,但需要特殊设备和环境,操作要求极为严格,不能进行药敏试验。培养法也有较高的特异性,而敏感性稍差,但方法简单再加上可快速做出药敏,配合临床用药,特别适合基层医院检测解脲支原体的好方法。

HBV-DNA和乙肝血清标志物的相关性研究

[目的]探讨乙肝血清学标志物(HBV-M)与乙肝病毒DNA间的关系。[方法]对320例血清标本,分别采用酶联免疫吸附试验(ELISA)和荧光定量聚合酶链反应(FQ-PCR)法检测。[结果]乙肝"大三阳"HBV-DNA阳性率为96.9%(95/98),"小三阳"HBV-DNA阳性率为59.9%(91/152,2=42.99,P<0.01)。HBeAg阳性标本中,HBV-DNA阳性率为96.2%(100/104);HBeAg阴性标本中,HBV-DNA阳性率为60.2%(130/216,2=44.93,P<0.01)。[结论]血清HBV-DNA能直接反映HBV的存在状态及感染性标志。在乙肝诊治中,不能仅凭检测乙肝病毒标志物,同时应做HBV-DNA定量测定。