Identification of enterotoxigenic Escherichia coli with synthetic alkaline phosphatase-conjugated oligonucleotide DNA probes

Abstract

Alkaline phosphatase-conjugated (AP) 26-base oligonucleotide DNA probes were compared with the same probes labeled with y_32p for the identification of heat-labile (LT) and heat-stable (ST) enterotoxigenic Escherichia coli (ETEC). The AP oligonucleotide probes were as sensitive as the radiolabeled (RL) probes in detecting LT and STA-2 target cell DNA, but the AP ST probe, which differed from STA-1 by two bases, was less sensitive than the RL probe in detecting STA-1 DNA (6.25 versus 0.78 ng). Of 94 ETEC that were identified with the RL probe, the AP probes detected 93% (28 of 30) of ST, 73% (25 of 34) of LT, and 67% (20 of 30) of LTST ETEC. When colony lysates of these ETEC were examined, the AP probes identified all 94 ETEC. In examinations of stool blots, the RL and AP probes were shown to have sensitivities of 71 and 59%, specificities of 91 and 86%, positive predictive values of 87 and 73%, and negative predictive values of 86 and 74%, respectively. AP oligonucleotide probes to detect ETEC were less sensitive in detecting ETEC by colony or stool blot hybridization than the RL probes but could be used by laboratories without access to radioisotopes to examine colony lysates.

Cloned polynucleotide and synthetic oligonucleotide probes used in colony hybridization are equally efficient in the identification of enterotoxigenic Escherichia coli

“…The colony hybridization assays described here demonstrate an acceptable sensitivity and specificity of oligonucleotide and polynucleotide probes in the detection of ETEC. When nonradioactive labeling techniques of gene probes (10,12,14,20) achieve the sensitivity of their radiolabeled ancestors, such methods will be increasingly applicable for clinical and epidemiological studies of diarrheal diseases. "Strains that were positive by hybridization with the LT and the ST probes are recorded in the LT and in the relevant ST row(s). "…”

“…Restriction endonucleasegenerated DNA fragments harboring specific toxin gene sequences, as well as synthetically produced oligonucleotides constructed to match such genes, have been used as probes for the identification of ETEC. In general, compared with the standard bioassays for detecting the phenotypic expression of these genes, the efficiency of colony hybridization with these two classes of probes has been satisfactory (16,(19)(20)(21). However, on a few occasions, the sensitivity has appeared to be suboptimal (3,4).…”

“…Colony hybridization assays have proved efficient in clinical and epidemiological studies of ETEC-induced diarrhea(16,(18)(19)(20). However, several apparent drawbacks have discouraged researchers to rely solely on this method, and therefore, time-consuming biological assays are often run in parallel.…”

Detection of enterotoxigenic Escherichia coli after polymerase chain reaction amplification with a thermostable DNA polymerase

“…Traditionally, nucleic acid hybridization has relied on radioactive labels for detection. Alternative, nonradioactive labels have been developed (7,8) and have been used successfully (1,12,13,17); however, the sensitivity of the assays requires large numbers of organisms for detection (12,13,17). Thus, the currently used hybridization assays are generally for culture confirmation rather than direct detection and identification.…”

Simplified and accurate nonradioactive polynucleotide gene probe assay for identification of enterotoxigenic Escherichia coli

“…Hybridization assays with nonradioactive ETEC probes have been described earlier (10,12,(17)(18)(19)(20). Tests with alkaline phosphatase-conjugated oligonucleotide ETEC probes show considerable variation with regard to sensitivity and specificity (12,(17)(18)(19)(20). These tests are mostly supplied in kits that include expensive reagents such as proteinase K. One of the assays did not require proteinase K but was only applicable for the detection of STaII (17).…”