Zhu Q, Lim C K, Chan Y N
Department of Microbiology, National University of Singapore.
J Appl Bacteriol. 1996 Mar;80(3):244-51. doi: 10.1111/j.1365-2672.1996.tb03216.x.
A rapid and sensitive method for detection of Salmonella typhi would help in preventing the spread of outbreaks and in clinical diagnosis. In order to develop unique PCR primers to detect Salm. typhi, ribosomal RNA genes from Salm. typhi (Rawlings) were cloned in pUC18. The resulting clone was confirmed by sequencing. The cloned DNA fragment contained the 5S, part of the 23S rRNA genes and the 5S-23S spacer region (EMBL/GenBank accession No. U04734). It was expected that the 5S-23S spacer region is divergent unlike the highly conserved 23S + 5S genes. This was confirmed by comparison with the rRNA gene sequences in the EMBL/GenBank database. A pair of PCR primers specific for Salm. typhi was obtained, based on this spacer region sequence. The specificity of this pair of primers was tested with 54 Salm. typhi strains (of 27 different phage types). All these Salm. typhi strains showed the positive 300 bp PCR product with this pair of primers. Six other Salmonella species as well as six other non-Salmonella bacteria were tested and none showed the 300 bp PCR product. The sensitivity of the detection level was 0.1 pg of pure Salm. typhi genomic DNA, or approximately 40 Salm. typhi cells in a spiked food sample. This pair of primers therefore has the potential for development into a diagnostic tool for the rapid diagnosis of typhoid fever.
一种快速灵敏的伤寒沙门氏菌检测方法将有助于预防疫情爆发的传播以及临床诊断。为了开发用于检测伤寒沙门氏菌的独特PCR引物，将伤寒沙门氏菌（罗林斯）的核糖体RNA基因克隆到pUC18中。通过测序确认所得克隆。克隆的DNA片段包含5S、部分23S rRNA基因和5S-23S间隔区（EMBL/GenBank登录号U04734）。预计5S-23S间隔区与高度保守的23S + 5S基因不同，具有差异性。通过与EMBL/GenBank数据库中的rRNA基因序列比较得以证实。基于该间隔区序列获得了一对针对伤寒沙门氏菌的PCR引物。用54株伤寒沙门氏菌菌株（27种不同噬菌体类型）测试了这对引物的特异性。所有这些伤寒沙门氏菌菌株用这对引物均显示出阳性的300 bp PCR产物。还测试了其他六种沙门氏菌以及六种其他非沙门氏菌细菌，均未显示出300 bp PCR产物。检测水平的灵敏度为0.1 pg纯伤寒沙门氏菌基因组DNA，或在加标食品样品中约40个伤寒沙门氏菌细胞。因此，这对引物有潜力开发成为用于伤寒热快速诊断的诊断工具。
