Lee C Y, Pan S F, Chen C H
Graduate Institute of Agricultural Chemistry, National Taiwan University, Taipei, Republic of China.
Appl Environ Microbiol. 1995 Apr;61(4):1311-7. doi: 10.1128/aem.61.4.1311-1317.1995.
The nucleotide sequence of pR72H cloned from Vibrio parahaemolyticus 93 was determined. We examined all V. parahaemolyticus gene sequences published in the GenBank-EMBL databases for homology and found that no other DNA sequence of V. parahaemolyticus was highly homologous to the sequence reported in this study. A pair of primers, VP33-VP32, derived from a pR72H fragment were selected to detect V. parahaemolyticus. The sensitivity of PCR detection for a pure culture of V. parahaemolyticus was 10 cells from crude bacterial lysates. Furthermore, a detection level of 2.6 fg, equivalent to 1 cell, was obtained by using purified chromosomal DNA as the template. The expected PCR products were obtained from all V. parahaemolyticus strains tested (n = 124), while no PCR amplicons were found in other vibrios or related genera (n = 50). High levels (10(6) to 10(10) CFU/ml) of Escherichia coli cells did not affect the PCR assay sensitivity. The presence of 10(8) V. parahaemolyticus cells or 10(9) E. coli cells in the PCR mixtures completely inhibited the PCR. When oyster samples were inoculated with V. parahaemolyticus 93 and cultured in tryptic soy broth containing 3% NaCl for 3 h at 35 degrees C, an initial sample inoculum level of 9.3 CFU/g was detected in a PCR assay with crude bacterial lysates. The PCR assay with enrichment culturing in salt polymyxin broth was compared with the conventional method for naturally contaminated shellfish and fish samples. We conclude that this PCR assay with enrichment culturing is a good alternative method for the detection of V. parahaemolyticus.
测定了从副溶血性弧菌93中克隆的pR72H的核苷酸序列。我们在GenBank-EMBL数据库中检查了所有已发表的副溶血性弧菌基因序列的同源性，发现没有其他副溶血性弧菌的DNA序列与本研究报道的序列高度同源。选择一对源自pR72H片段的引物VP33-VP32来检测副溶血性弧菌。对于副溶血性弧菌纯培养物，PCR检测的灵敏度为来自粗细菌裂解物的10个细胞。此外，以纯化的染色体DNA为模板，检测水平达到2.6 fg，相当于1个细胞。从所有测试的副溶血性弧菌菌株（n = 124）中均获得了预期的PCR产物，而在其他弧菌或相关属（n = 50）中未发现PCR扩增产物。高水平（10⁶至10¹⁰ CFU/ml）的大肠杆菌细胞不影响PCR检测的灵敏度。PCR混合物中存在10⁸个副溶血性弧菌细胞或10⁹个大肠杆菌细胞会完全抑制PCR。当用副溶血性弧菌93接种牡蛎样品，并在含3% NaCl的胰蛋白胨大豆肉汤中于35℃培养3小时后，用粗细菌裂解物进行PCR检测时，初始样品接种水平为9.3 CFU/g。将盐多粘菌素肉汤富集培养的PCR检测方法与天然污染的贝类和鱼类样品的传统方法进行了比较。我们得出结论，这种富集培养的PCR检测方法是检测副溶血性弧菌的一种很好的替代方法。
