Taylor D E, Hiratsuka K
Department of Medical Microbiology, University of Alberta, Edmonton, Canada.
Mol Cell Probes. 1990 Aug;4(4):261-71. doi: 10.1016/0890-8508(90)90018-u.
DNA probes specific for C. jejuni (pDT1720 containing a 1475 base pair fragment) and for C. jejuni and C. coli (pDT1719 containing a 1845 base pair fragment) were isolated from a bacteriophage lambda gt11 genomic library of C. jejuni, using antiserum prepared against a 46 kDa major outer membrane protein of C. jejuni. The two probe-fragments had different restriction maps and were only moderately related by DNA hybridization analysis. A non-radioactive labelling kit which consisted of alkaline phosphatase conjugated anti-digoxigenin antiserum and 5-bromo-4-chloro-3-indoyl phosphate with nitroblue tetrazolium as the colour substrate, which gives a purple colour for positive hybridization, was used to test 140 stool specimens, 70 of which were culture positive and 70 of which wer culture negative for Campylobacter spp. The pDT1720 fragment (C. jejuni probe) could detect a minimum of 1 x 10(5) C. jejuni cells on filters, whereas the pDT1719 fragment (C. coli probe) was 100-fold less sensitive. The C. jejuni probe demonstrated a sensitivity of 93% with culture positive stool samples, however, 15% of culture negative samples were also recorded as positive using this non-radioactive DNA probe.
使用针对空肠弯曲菌46 kDa主要外膜蛋白制备的抗血清，从空肠弯曲菌的λ噬菌体gt11基因组文库中分离出对空肠弯曲菌特异的DNA探针（含1475个碱基对片段的pDT1720）以及对空肠弯曲菌和结肠弯曲菌特异的DNA探针（含1845个碱基对片段的pDT1719）。这两个探针片段具有不同的限制性图谱，通过DNA杂交分析显示它们仅有中度相关性。一种非放射性标记试剂盒，由碱性磷酸酶偶联的抗地高辛抗血清以及以硝基蓝四唑为显色底物的5-溴-4-氯-3-吲哚磷酸组成，阳性杂交时呈紫色，用于检测140份粪便标本，其中70份弯曲菌属培养阳性，70份弯曲菌属培养阴性。pDT1720片段（空肠弯曲菌探针）在滤膜上能检测到至少1×10⁵个空肠弯曲菌细胞，而pDT1719片段（结肠弯曲菌探针）的灵敏度低100倍。空肠弯曲菌探针在培养阳性的粪便样本中显示出93%的灵敏度，然而，使用这种非放射性DNA探针时，15%的培养阴性样本也被记录为阳性。
