免疫PCR技术检测大肠埃希菌O157:H7的实验研究

目的通过对大肠埃希菌O157：H7阳性菌液和对照菌检测,评估免疫PCR技术的灵敏度和特异性。方法将大肠埃希菌O157：H7阳性菌液倍比稀释得到108~101CFU/ml菌悬液,用免疫PCR技术和ELISA进行检测并观察灵敏度。同时检测对照菌液观察免疫PCR技术特异性。结果免疫PCR检测O157：H7菌液最低浓度为10 CFU/ml时,检测灵敏度与ELISA相比较提高了1000倍,对照菌检测均为阴性。结论免疫PCR技术检测具有较高的灵敏度和特异性,可以有效提高大肠埃希菌O157：H7的确诊率,降低漏诊率。

噬菌体免疫PCR技术及其在真菌毒素检测中的应用

噬菌体免疫PCR(Phage-mediated immuno-polymerase chain reaction,PD-IPCR)技术融合了噬菌体展示技术、特异性抗原-抗体和PCR技术,是三者相结合而派生出来的一项新分析技术。本文介绍了PD-IPCR技术的发展历程、技术原理及类型和技术流程中的关键实验方法,列举了一些噬菌体免疫PCR技术及其在真菌毒素检测中的应用,并结合PD-IPCR技术特点,分析了PD-IPCR技术在真菌毒素混合污染同步检测和污染预警研究等方向上的重要应用前景。

免疫PCR技术的现状和展望

免疫PCR是近年出现的一种将免疫测定和PCR技术结合起来的新的抗原测定技术，具有极高的灵敏度．本文对其基本原理和方法作了较为详尽的介绍。

Quantification of Simian Immunodeficiency Virus by SYBR Green RT-PCR Technique

Plasma viral RNA load is widely accepted as the most relevant parameter to assess the status and progression of Simian immunodeficiency virus （SIV） infections. To accurately measure RNA levels of the virus, a one-step fluorescent quantitative assay was established based on the SYBR green Real-time reverse transcription-polymerase chain reaction （RT-PCR）. The lower detection limit of the assay was 10 copies per reaction for the virus. This method was successfully applied to quantify SIVmac251 and SIVmac239 viruses produced in CEMx174 cells. Additionally, the performance of the SYBR green RT-PCR was assessed in a SIVmac251 infected rhesus macaque. The result demonstrated that the method could detect as little as 215 copies per milliliter of plasma and the dynamic pattern of viral load was highly consistent with previous results. With regard to convenience, sensitivity and accuracy our assay represents a realistic alternative to both branched-chain DNA （b-DNA） assays or real-time PCR assays based on TaqMan probes.

Study on the Correlation between the Expression of Angiogenic Factor VEGF and Microsatellite Instability in Gastric Adenocarcinoma

Objective: To investigate the correlation between the microsatellite instability (MSI) and the expression of vascular endothelial growth factor (VEGF) in gastric adenocarcinoma. Methods: PCR SSCP method was used to detect MSI of thirty cases with gastric adenocarcinoma at five loci in each patient. Expression of VEGF was examined by the method of immunohistochemistry. Results: The positive of MSI was in 13 patients out of 30 patients (43.4%) in our study. Positive VEGF Immunostaining was detected in 18 patients (60.0%). VEGF was decreased in microsatellite instability high (MSI H) gastric adenocarcinoma. Conclusion: MSI H and microsatellite stable (MSS) gastric adenocarcinoma may follow a different pathway of angiogenesis. The low frequency of VEGF expression among MSI H cancer may partially explain why these cancers are less aggressive.

The quantitative expressions of lymphangiogenic factors and metastasis in human colorectal cancers

Objective： The purpose of this paper was to study the expression levels of newly described lymphatic endothelial markers – LYVE-1, Prox-1, podoplanin and 5’-nucleotidase, and their correlation with metastasis of human colorectal cancers. Methods： Tumor and corresponding tumor-side normal tissue samples were obtained from resected specimens immediately after operation. Expression level of each factor was determined by quantitative real-time PCR （RT-PCR） and Western blot technique. Results： Expression levels of lymphatic endothelial markers LYVE-1, Prox-1, podoplanin and 5’-nucleotidase were significantly different in tumor and tumor-side normal groups. Expression levels of Prox-1 and podoplanin were higher in patients with positive lymph node metastasis than those without metastasis. LYVE-1, but not 5’-nucleotidase expression level was higher in both cancer and normal groups. Conclusion： These results indicate that combined quantitative analysis of lymphangiogenic markers LYVE-1, Prox-1 and podoplanin in colorectal cancer specimens may be useful in predicting metastasis of colorectal cancer to regional lymph nodes. However, the role of 5’-nucleotidase in predicting metastasis of colorectal cancer still remains to be further analyzed.

Farnesoid X receptor expression is reduced in human hepatocellular carcinoma

Farnesoid X receptor (FXR, NR1H4) is a member of nuclear hormone receptor superfamily. Previously studies showed that FXR-/- mice spontaneously developed liver tumors when they aged, however, the relevance of which to human hepatocellular carcinoma (HCC) is unclear. The aim of this study is to observe whether FXR expression is also downregulated in HCC and discuss the mechanism of the reduced FXR expression in HCC. Expression of FXR and small heterodimer partner (SHP) was measured by real-time PCR and immunohistochemical technique. Effect of pro-inflammatory cytokines on expression of FXR and its promoter activity were determined in primary hepatocytes or HepG2 and Huh7 cell lines. Our results showed that expression of FXR and its target gene SHP in human HCC was strongly downregulated compared to the normal liver tissues. In addition, pro-inflammatory cytokines were able to decrease FXR expression by inhibiting the FXR promoter activity. In conclusion this work demonstrates FXR expression is strongly downregulated in human HCC, which may be caused by decreased FXR promoter activity, suggesting a potential role of FXR in human HCC development.

乙型肝炎患者HBV DNA荧光定量检测临床分析

目的 了解乙肝患者HBVDNA含量及HBVDNA阳性患者用药前后的疗效观察。方法应用荧光PCR扩增病毒核酸免疫技术定量检测 6 5 0 0例血清标本的HBVDNA含量。结果 荧光定量PCR的特点与定性PCR主要区别在于荧光PCR即时反映扩增过程 ,采用内标法全封闭减少污染 ,足够灵敏的自动化仪器对荧光的收集和分析能达到对原始模板量定量检测。结论 用此方法可较准确、及时地为临床提供科学的诊断依据 ,并对HBVDNA阳性病人用药前后提供可靠数据。