Direct detection of Escherichia coli Shiga-like toxin genes in primary fecal cultures by polymerase chain reaction.

A single pair of oligonucleotide primers was used for polymerase chain reaction amplification of a 212- or 215-bp region of Escherichia coli Shiga-like toxin (SLT) genes from crude fecal culture extracts. Genes were typed by hybridization of the polymerase chain reaction products to SLT-I- or SLT-II-specific oligonucleotide probes. The procedure was capable of detecting fewer than 10 SLT-producing E. coli organisms per ml of culture against a background of more than 10(9) other organisms per ml and provides a rapid and sensitive means of screening primary fecal cultures for the presence of such strains. When this procedure was used to test primary cultures from gut contents or feces from various patient groups, including apparently healthy infants, approximately half of all samples yielded positive results for SLT-I and/or SLT-II sequences.     

Single primer pair for amplifying segments of distinct Shiga-like-toxin genes by polymerase chain reaction.

By using a single synthetic oligonucleotide primer pair in the polymerase chain reaction, we amplified specific Shiga-like-toxin (SLT) gene segments from DNAs of 20 clinical Escherichia coli isolates, irrespective of whether they produce SLT-I, SLT-II, or heretofore uncategorized SLTs. These segments were not detectable in any of 20 nontoxigenic E. coli strains. The primers deduced from a conserved region among SLT genes are so-called degenerate-sequence primers; i.e., they contain intentionally introduced sequence ambiguities to overcome minor sequence variations within different SLT genes. In direct gel hybridization with genomic DNA, both primers recognized SLT-I and SLT-II DNA sequences. Amplified sequences of target DNA obtained by polymerase chain reaction were visualized after gel electrophoresis by ethidium bromide staining, and definitive identification of the amplification product as an SLT gene segment was achieved by hybridization to SLT-I- and SLT-II-specific 20-base oligonucleotide probes complementary to a portion of the amplified sequences but not to the primers. The detecting oligonucleotide probes shared only 30% base homology and were shown to recognize specifically SLT-I or SLT-II sequences within genomic DNA. Moreover, they were used to distinguish whether the amplified sequence originated from SLT-I or SLT-II genes. The PCR system with the primers described here is a powerful technique to amplify SLT sequences in E. coli strains that produce serologically distinct SLTs and will facilitate identification of these pathogens, particularly among a multitude of nonpathogenic E. coli strains.     

Detection of diarrheagenic Escherichia coli by multiplex PCR

Background: Diarrheagenic E.coli (DEC) are an important cause of childhood diarrhea.Identification of DEC strains needs to detect factors that determine the virulence of these organisms. There is not much data regarding the importance of DEC as a cause of diarrhea in children in India.The prevalence of DEC in children belowfive years with and without diarrhea was studied using two multiplex PCR assays. Materials and Methods: Two multiplex polymerase chain reaction assays were used to detect genes of five types of DEC.The targets selected for each category were eae and bfpA (bundle-forming pilus) forEnteropathogenic E.coli (EPEC), hlyA for Enterohemorrhagic E.coli (EHEC), elt and stla for Enterotoxigenic E.coli (ETEC), CVD432 for Enteroaggregative E.coli (EAEC) and ial for Enteroinvasive E.coli (EIEC). Results: In 200 children with diarrhea 52 (26%) DEC infections were found. Among 100 controls 8 (8%) DEC infections were found. EAEC was the most common DEC by multiplex PCR both in cases (26, 13%)and controls (5,5%), followed byEPEC seen in 16% cases and 3% controls. ETEC and EIEC were found in 7 (3.5%) and 3 (1.5%) of the diarrheal cases. EIEC and ETEC were not detected in the control cases. EHEC was not isolated from either the diarrheal or control cases. Conclusion: DEC strains are a significant cause of diarrhea in children. The two Multiplex PCR assays can be used for the detection of DEC in routine diagnostic laboratories. These assays are specific and sensitive for the rapid detection of DEC. EAEC was the most frequent pathotype in the population under study.     

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

The direct identification of enterotoxigenic Escherichia coli from clinical specimens was examined by using the polymerase chain reaction (PCR) for amplifying the heat-labile toxin (LT) gene. Two synthetic primers, each of which was 20 bases in length, were used with the thermostable DNA polymerase from Thermus aquaticus to amplify the LT gene. The amplified PCR products were detected by either gel electrophoresis or hybridization to a 24-base synthetic oligonucleotide probe conjugated to alkaline phosphatase. The PCR method detected LT-positive bacteria but did not react with E. coli producing the heat-stable toxin, enteroinvasive E. coli, Salmonella typhi, Salmonella typhimurium, or Shigella dysenteriae. By the PCR method, a single bacterium could be detected following 30 cycles of amplification. The T. aquaticus DNA polymerase was inhibited by more than 10(3) organisms in the amplification reaction mixture. A group of 40 clinical specimens consisting of 16 LT bioassay-positive and 24 LT bioassay-negative stool specimens were tested by PCR for the presence of toxigenic E. coli. The total DNA from 100 microliters of stool specimen was extracted and partially purified with a commercially available ion-exchange column. All 16 of the bioassay-positive stool specimens were positive by PCR. In addition, one stool specimen which was bioassay negative for LT but positive for LT in a previous hybridization assay with a different LT probe was also positive by PCR. This may indicate that the LT gene is present but either is not expressed or is expressed below detectable levels. Amplification of specific DNA sequences by PCR provides a highly sensitive and specific tool for the detection of pathogenic microorganisms directly from clinical specimens without the need for prior isolation. This technique may find wide application in the detection of other organisms in addition to enterotoxigenic E.coli.     

Clinical and genetic aspects of Shiga-like toxin production in traditional enteropathogenic Escherichia coli

Cell culture tests, DNA colony blot hybridization and polymerase chain reaction were used to examine classical enteropathogenic Escherichia coli (EPEC) for the presence of Shiga-like toxin (SLT). Fifteen of 155 strains from West Germany, originally identified as EPEC on the basis of serotyping, were shown to harbor either SLT-I or SLT-II genes. All strains that hybridized with the 20-base oligonucleotide probes which are complementary to slt-IA or slt-IIA sequences derived from the genomic DNA of enterohemorrhagic E. coli O157:H7 strain 933 produced moderate or high levels of cytotoxin in Vero and HeLa cell assays. Four additional strains of low to moderate cytotoxicity did not hybridize with either probe. Five different serogroups producing SLTs were identified: O26, O55, O111, O119 and O128. All three SLT-positive E. coli O26:H11 and four of five E. coli O111:H- isolates hybridized with a 3.4 kilobase fragment (CVD 419 probe) derived from the 60-megadalton plasmid of EHEC O157:H7. Seven of the 15 SLT-gene positive strains were associated with bloody diarrhea, six isolates were from patients with hemolytic uremic syndrome (HUS). Based on their clinical, epidemiological, pathogenic and genetic features SLT-producing E. coli among classical EPEC mimic enterohemorrhagic E. coli O157:H7 and might be considered as EHEC.     

Rapid and specific detection of verotoxin genes in Escherichia coli by the polymerase chain reaction.

A set of four synthetic oligonucleotide probes derived from sequences of the VT1 (Shiga-like toxin I [SLT-I]) and VT2 (SLT-II) genes were used in a polymerase chain reaction (PCR) amplification procedure to detect these genes in some enteric pathogens. A total of 40 verotoxin-producing Escherichia coli strains and 43 isolates of other recognized enteric pathogens were studied. PCR amplification products identifying the VT1 and VT2 gene sequences were observed only in nucleic acid extracted from strains found to be VT positive in traditional tissue culture assays. Template nucleic acid extracted from other gram-negative bacteria was found to be negative with the exception of five isolates of Shigella dysenteriae type 1 in which good amplification with the VT1 probe was observed. The oligonucleotide probes clearly distinguished VT1 and VT2 strains of E. coli and did not give specific amplification with nucleic acid from VTe (a SLT-II variant)-producing E. coli. VT1 or VT2 genes or both were not detected in E. coli K-12 strain C600 or HB101 or in strains known to express other virulence factors, such as enterotoxins, adhesins, hemolysins, or unrelated cytotoxins. The sensitivity of the PCR procedure for detection of both VT1 and VT2 genes was determined to be 1 ng of total nucleic acid. Furthermore, the VT1 gene was easily detected when only 100 pg of nucleic acid was used as the template in the PCR procedure.     

Detection of genes in feces by booster polymerase chain reaction.

A 321-bp fragment intragenic to the gene ereA carried by Escherichia coli BM2195 was used as a model target to study the conditions under which DNA amplification by booster polymerase chain reaction can be used to detect specific bacterial DNA sequences in fecal specimens. When target E. coli cells were mixed with 41 freshly obtained fecal specimens, the polymerase chain reaction detection limit varied from 4.5 to 7.1 log CFU/g of feces, depending on the individual fecal specimen used to prepare the test sample. These variations were not statistically related to the sex or age of the subject from whom the specimen was obtained. After storage of the samples for 4 weeks at room temperature on swabs or filter papers, no loss in sensitivity was observed.     

Detection of varicella-zoster virus (VZV) DNA in clinical samples from patients with VZV by the polymerase chain reaction.

A polymerase chain reaction system for the detection of varicella-zoster virus was established. Of 25 nucleotides, 4 oligonucleotide pairs (regions of thymidine kinase, thymidylate synthetase, glycoprotein I, and immediate early gene) were synthesized. The first three oligonucleotide pairs could be used as primers on the basis of specific DNA amplification. Varicella-zoster virus DNA was amplified by this polymerase chain reaction system in 20 of 20 vesicle samples, 5 of 6 crusts, and 12 of 13 throat swabs collected from patients with clinical varicella.     

Detection ofChlamydia trachomatis in clinical specimens by the polymerase chain reaction

Sequences derived from the endogenous plasmid ofChlamydia trachomatis and from the genes coding for ribosomal 16S RNA ofChlamydia psittaci were used as primers and oligonucleotide probes for detection of chlamydiae by the polymerase chain reaction. The endogenous plasmid primers generated specific amplified products of 517 bp with all knownChlamydia trachomatis serovars. No specific products ofChlamydia psittaci andChlamydia pneumoniae could be detected using these primers. With the rRNA primers specific amplified products of 208 bp were generated withChlamydia psittaci, Chlamydia trachomatis andChlamydia pneumoniae. No specific amplified products were detected with DNA isolated from a variety of microorganisms from the urogenital and the respiratory tract. Of 156 clinical specimens used for evaluation of the polymerase chain reaction, 26 were found to be positive forChlamydia trachomatis on culture. All 26 culture positive samples were also found to be positive forChalmydia trachomatis DNA by the polymerase chain reaction with both primer sets. Two culture negative samples were also found to be positive by this technique. The polymerase chain reaction thus seems to be a sensitive and reliable method for detection ofChlamydia trachomatis.     

Detection and strain differentiation of Chlamydia psittaci mediated by a two-step polymerase chain reaction.

Specific and sensitive amplification of major outer membrane protein (MOMP) gene DNA sequences of Chlamydia psittaci was achieved in a two-step polymerase chain reaction. First, oligonucleotide primers specific for 5' and 3' nontranslated regulatory regions of the MOMP gene were used in a polymerase chain reaction to amplify a DNA fragment of approximately 1,400 bp. A portion of this DNA fragment was amplified in a second reaction using a degenerate oligonucleotide primer specific for a DNA sequence contained within the 1,400-bp DNA fragment and one of the first-step primers. This method detected 10 cognate chlamydial genomes. C. psittaci MOMP genes from two avian strains and from mammalian serovars 1, 7, and 8 were amplified and analyzed by restriction endonuclease digestion. MOMP genes from mammalian serovars 2 through 6 and 9 and from strains of C. trachomatis and C. pneumoniae could not be amplified. Restriction endonuclease analysis with HaeIII indicated a close relationship between C. psittaci strains of avian and mammalian serovar 1 lineage, while those of mammalian serovars 7 and 8 exhibited distinct restriction patterns. DNA sequences corresponding to the mammalian serovar 1-wild type parakeet MOMP genotype of C. psittaci were detected in two of seven milk samples from cases of bovine mastitis.     

Rapid and specific detection of the pap, afa, and sfa adhesin-encoding operons in uropathogenic Escherichia coli strains by polymerase chain reaction.

Adhesin-encoding operons (pap, sfa/foc, and afa) have been shown to be prevalent in Escherichia coli strains associated with urinary tract infections. A quick and sensitive assay to identify these operons was developed by using the polymerase chain reaction (PCR). Three pairs of 25-mer primers were defined from the sequences of the DNA fragments used as probes in hybridization studies to identify each of the three operons, and the six primers were used together in a single reaction of amplification. To validate the PCR approach for detection of adhesin-encoding operons among clinical isolates, we investigated a collection of 97 E. coli isolates with the following characteristics: all isolates originated from the urine of patients with pyelonephritis, and the adhesin responsible for specific binding of the isolates to uroepithelial cells was previously characterized by phenotypic assays, as well as genotypic tests based on hybridization. There was a perfect correlation between the results obtained with the PCR approach and those previously obtained by using DNA probes. These results indicate that the PCR method, which is highly specific and easier to perform than the hybridization method, is a powerful genotypic assay for detection of adhesin-encoding operons. Thus, this assay can be recommended for clinical use to detect virulent urinary E. coli strains, as well as for epidemiological studies.     

Detection of diarrheagenic Escherichia coli by use of melting-curve analysis and real-time multiplex PCR

Diarrheagenic Escherichia coli strains are important causes of diarrhea in children from the developing world and are now being recognized as emerging enteropathogens in the developed world. Current methods of detection are too expensive and labor-intensive for routine detection of these organisms to be practical. We developed a real-time fluorescence-based multiplex PCR for the detection of all six of the currently recognized classes of diarrheagenic E. coli. The primers were designed to specifically amplify eight different virulence genes in the same reaction: aggR for enteroaggregative E. coli, stIa/stIb and lt for enterotoxigenic E. coli, eaeA for enteropathogenic E. coli and Shiga toxin-producing E. coli (STEC), stx(1) and stx(2) for STEC, ipaH for enteroinvasive E. coli, and daaD for diffusely adherent E. coli (DAEC). Eighty-nine of ninety diarrheagenic E. coli and 36/36 nonpathogenic E. coli strains were correctly identified using this approach (specificity, 1.00; sensitivity, 0.99). The single false negative was a DAEC strain. The total time between preparation of DNA from E. coli colonies on agar plates and completion of PCR and melting-curve analysis was less than 90 min. The cost of materials was low. Melting-point analysis of real-time multiplex PCR is a rapid, sensitive, specific, and inexpensive method for detection of diarrheagenic E. coli.     

Elimination of contaminating DNA within polymerase chain reaction reagents: implications for a general approach to detection of uncultured pathogens.

Analysis based on comparisons of 16S rRNA sequences provides a rapid and reliable approach to identifying human pathogens. By directing oligonucleotide primers at sequences conserved throughout the eubacterial kingdom, bacterial 16S ribosomal DNA sequences of virtually any member of the eubacterial kingdom can be amplified by polymerase chain reaction and subsequently analyzed by sequence determination. Indeed, automated systems for broad-range amplification, sequencing, and data analysis are now feasible and may form the basis of the next generation of automated microbial identification systems. However, identification of pathogens by this strategy is hampered by the frequent contamination of reagents used for the amplification reaction, in particular Taq polymerase, with exogenous bacterial DNA. Here, we describe detailed investigations on the use of 8-methoxypsoralen and long-wave UV light to eliminate contaminating DNA in polymerase chain reaction reagents. The clinical utility of the developed procedure was demonstrated in a case of paucibacillary osteomyelitis, for which no specific bacterial agent had been cultured.     

Development of oligonucleotide primers and probes against structural and regulatory genes of human immunodeficiency virus type 1 (HIV-1) and their use for amplification of HIV-1 provirus by using polymerase chain reaction.

The polymerase chain reaction is a powerful technique for amplifying a few copies of double-stranded genetic material to millions of copies in a few hours. The sensitivity and specificity of the polymerase chain reaction technique depend to some extent on the nucleotide sequences of the oligonucleotide primer pair used in the amplification. We report new oligonucleotide primers and probes which can be used for the amplification and detection of human immunodeficiency virus type 1 provirus sequences of not only structural but also regulatory genes. These primers are very sensitive and specific and can be used for the detection of African and North American strains of human immunodeficiency virus type 1.     

Detection of Shiga toxin-producing Shigella dysenteriae type 1 and Escherichia coli by using polymerase chain reaction with incorporation of digoxigenin-11-dUTP.

A technique has been developed for the detection of Shiga toxin- and Shiga-like toxin type I (ShT/SLT-I)-producing Shigella dysenteriae type 1 and Escherichia coli by using the polymerase chain reaction with the incorporation of digoxigenin-11-dUTP. Target DNA liberated from whole cells was amplified, using primer pairs homologous to the A-subunit genes of ShT/SLT-I. The TTP analog digoxigenin-11-dUTP was incorporated into the reaction mixture, permitting nonradioactive labeling of the amplified DNA. The labeled polymerase chain reaction products were hybridized to specific gene sequences immobilized on a nitrocellulose membrane and detected by using an alkaline phosphatase-conjugated antibody to digoxigenin and the enzyme substrates. Toxin-producing strains of E. coli and S. dysenteriae type 1 were identified as colored spots on the membrane. Because this technique does not require DNA purification, gel electrophoresis, or radioactive DNA probes, it is suitable for the clinical detection of ShT/SLT-I-producing strains of S. dysenteriae type 1 and E. coli.     

Rapid and safe determination of human apolipoprotein E genotypes by miniaturised SDS-PAGE in non-insulin dependent diabetes mellitus.

AIMS--To present a non-isotopic procedure for the analysis of the different apolipoprotein E genotypes in normal subjects and patients with non-insulin dependent diabetes mellitus. METHODS--Apolipoprotein E genotypes were detected following polymerase chain reaction and miniaturised sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) (PhastSystem Pharmacia). RESULTS--The time taken from extraction of DNA from 6 microliters whole blood to the final result was eight hours. The allele frequencies in patients with diabetes mellitus in our series were similar to those found in the control subjects. CONCLUSIONS--SDS-PAGE is rapid, reliable and safe with few drawbacks, can be used as a routine procedure and may easily replace apolipoprotein E phenotyping.     

Gene probes for identification of the botulinal neurotoxin gene and specific identification of neurotoxin types B, E, and F.

A polymerase chain reaction method was developed for the specific detection of the botulinum neurotoxin (BoNT) gene of Clostridium botulinum. Degenerate oligonucleotide primers, designed from the nucleotide sequence of the heavy chain of the BoNT gene, amplified a specific fragment of approximately 1.1 kb from strains of C. botulinum toxin types A, B, E, F, and G and neurotoxin-producing strains of Clostridium barati and Clostridium butyricum, but no fragment was obtained from nontoxigenic strains. The fragments amplified from several strains of C. botulinum types B, E, and F were cloned in Escherichia coli and their nucleotide sequences were determined. Sequences within this region were used to design oligonucleotide probes specific for BoNT type B (BoNT/B), BoNT/E, and BoNT/F genes. An additional probe was designed for the detection of the BoNT/F gene of C. barati, which differed in sequence from BoNT/F genes of both proteolytic and nonproteolytic strains of C. botulinum.     

聚合酶链式反应快速检测E型肉毒神经毒素基因

目的Ｅ型肉毒中毒是人类肉毒中毒的主要型别之一，本试验旨在建立用于Ｅ型肉毒梭菌鉴定的ＰＣＲ方法。方法用人工合成的寡核苷酸引物扩增Ｅ型肉毒神经毒素基因的一段３６８ｂｐ的ＤＮＡ片段，快速检测Ｅ型肉毒神经毒素基因，对梭状芽胞杆菌属的４０株保藏菌株及３３份土壤标本进行了鉴定，用Ｅ型肉毒梭菌ＣＭＣＣ（Ｂ）６４５０１对其灵敏度进行了检查。结果从所有Ｅ型神经毒素原性菌株或标本均能扩增出目的片段，且能用特定的限制性内切酶切成相应的片段。其它菌株均未能扩增出目的片段。灵敏度试验可从３０个菌体扩增出目的片段。结论此方法用于Ｅ型神经毒素原性梭菌的鉴定具有较高的灵敏度及特异性。     

Enterotoxigenic Escherichia coli in Central Australia: diagnosis using cloned and synthetic nucleic acid probes

Feces from 169 children admitted with diarrhea to Alice Springs Hospital, were screened for enterotoxigenic E. coli (ETEC) using specific DNA probes. E. coli which hybridized with probes for ST-P, ST-H and LT were confirmed by bioassay for toxin production. Fifty children were shown to excrete ETEC. The probes for LT correlated well with bioassay; however, probes for ST-H and ST-P hybridized with more E. coli than were shown to produce toxin by bioassay. When probing for ST was repeated using a synthetic oligonucleotide probe, only those specimens which were bioassay-positive hybridized with the probe.