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目的以普氏立克次体为研究对象，建立一种定量测定胞内微生物繁殖的方法。方法从立克次体感染细胞提取总ＲＮＡ为样品或用硫氰酸胍裂解感染细胞，以裂解物为样品，用３２Ｐ标记的１６ＳｒＲＮＡ特异探针做ＲＮＡ酶保护分析。根据探针分子结合数，计算出检测到的靶序列分子数，反映胞内微生物的繁殖量。结果从０．５μｌ直接裂解物可检测到３．９４×１０４个１６ＳｒＲＮＡ分子；从６ｐｇ的总ＲＮＡ中可检测到５．８２×１０４个１６ＳｒＲＮＡ分子。用本法试测立克次体在宿主细胞内的生长曲线，只表现出迟缓期及对数生长期，而无稳定期及衰退期。结论用特异性探针通过ＲＮＡ酶保护分析法可以定量测定胞内微生物在宿主细胞内的繁殖量。由本法测定到的普氏立克次体生长曲线表明难以沿用细菌的生长曲线来描述胞内微生物在宿主细胞内的生长过程
OBJECTIVE To establish a method for the quantitative determination of intracellular microbial growth using the Platts rickettsia as research object. Methods Total RNA was extracted from Rickettsia infected cells or infected with guanidinium thiocyanate to lyse the lysate as a sample and 32P-labeled 16S rRNA-specific probe was used to perform RNase protection analysis. According to the number of probe molecules binding, calculate the number of target molecules detected, reflecting the intracellular multiplication of microorganisms. Results 3.94 × 10 4 16S rRNA molecules were detected from 0.5 μl direct lysate and 5.82 × 10 4 16S rRNA molecules were detectable from 6 pg total RNA. Using this method to test the growth curve of rickettsia in host cells, only the lag phase and logarithmic growth phase, but no stable period and decay period. Conclusion The specific probe can be used to quantitatively determine the intracellular microbial multiplication in the host cell by RNase protection assay. The growth curve of Plattsia rickettsii determined by this method shows that it is difficult to describe the growth process of intracellular microorganisms in host cells following the growth curve of bacteria