定量检测幽门螺杆菌细胞毒素相关蛋白基因A方法的建立

目的建立定量检测幽门螺杆菌（Ｈｐ）细胞毒素相关蛋白基因Ａ（ｃａｇＡ）的方法。方法以含ＨｐｃａｇＡ片段的质粒（ｐＭＣ３）作为外参照品，在微孔板中对聚合酶链反应（ＰＣＲ）扩增ｃａｇＡ的产物进行辣根过氧化物酶标记探针的杂交及酶促显色以定量检测ｃａｇＡ阳性Ｈｐ。结果当ＰＣＲ反应体系中原始模板为１～１０５拷贝，循环次数小于或等于２５时，原始模板以相对固定的指数形式增加，在该范围内适宜定量分析。通过对２０份已知Ｈｐ菌株ｃａｇＡ的检测，符合率达１００％，观察结果的敏感度较电泳法提高了２００倍。定量检测不同胃黏膜组织中ｃａｇＡ阳性Ｈｐ，检出底线为６拷贝／毫克组织。结论本方法特异性强，敏感度高，对研究Ｈｐ与消化道疾病的关系及建立定量检测其他微生物的方法均有实用价值

Quantitative assay for detecting cytotoxin associated gene A of Helicobacter pylori

Objective To create a quantitative assay for cytotoxin associated gene A(cagA) positive Helicobacter pylori (Hp). Methods With plasmid pMC3, which contains cagA of Hp, being used as the external standard, one strand of the PCR product of cagA was hybridized with a horseradish peroxidase (HRP) coupled single strand probe in microplate wells for colorimetric and quantitative detection of cagA positive Hp. Results When the templates in the PCR system were over a range from 1 to 10 5 copies per microliter and the cycles of PCR were 25 or less than 25, the amplicon increased with a relatively constant exponential rate that was very suitabe for quantitation of the original templates. The coincident rate was 100% when 20 Hp strains with known characters were detected and the sensitivity of detection was 200 times that of electrophoresis. The copies of cagA positive Hp were also determined in several tissues with different histological features. The detection limit was 6 copies per microgram. Conclusion This is a specific, sensitive and quantitative assay for Hp and has significant rules in the relationship study of Hp with digestive tract diseases and in quantitative detection of other microorganisms.