Insertion element IS3-based PCR method for subtyping Escherichia coli O157:H7

An Escherichia coli O157:H7 subtyping method based on PCR amplification of variable DNA sequences between the repetitive element IS3 was developed. Template DNA was prepared by boiling cells in Chelex. Two separate IS3 PCR amplifications were performed for each isolate: one with a single primer (primer IS3A) and one with two primers (primers IS3A and IS3B). The IS3 PCR subtyping method was applied to 35 epidemiologically related and unrelated E. coli O157:H7 isolates that had been previously characterized by pulsed-field gel electrophoresis (PFGE). PFGE identified 25 different subtypes (difference of one or more bands). PCR with single primer IS3A and primer pair IS3A-IS3B identified 6 and 14 different subtypes, respectively. By combining the results of the two PCR amplifications, 15 different IS3 PCR subtypes were identified. While not as sensitive as PFGE, IS3 PCR subtyping grouped all outbreak-related isolates. IS3 PCR banding patterns were reproducible between amplifications and between subcultures. IS3 PCR could serve as a simple, rapid screening method for the identification of unrelated E. coli O157:H7 isolates.     

A nested PCR for detection of North American isolates of bluetongue virus based on NS1 genome sequence analysis of BTV-17

A nested polymerase chain reaction (PCR)-based assay, for detection of bluetongue virus (BTV) ribonucleic acid in cell culture and tissue samples, was developed. Two pairs of oligonucleotide primers (BTV1 and BTV4 and BTV2 and BTV3), selected from non-structural protein 1 (NS1) gene of BTV-17, were used for the nested PCR in two amplification steps. First a 826-bp product was amplified using an outer primer pair BTV1 and BTV4. The second amplification, using nested or internal primer pair BTV2 and BTV3, produced a 517-bp PCR product. RNA from North American prototype serotypes 2, 10, 11, 13 and 17, propagated in cell cultures, were detected by this nested PCR-based assay. The nested primers BTV2 and BTV3 increased the sensitivity of the BTV PCR assay, and as little as 0.1 fg of BTV RNA (equivalent to 5 viral particles) could be detected. Amplification products were not detected when the PCR-based assay was applied to RNA from a closely related orbivirus, epizootic hemorrhagic disease virus (EHDV) prototype serotypes 1 and 2; total nucleic acid extracts from uninfected BHK-21 cells; or whole blood from calves and deer that were BTV-seronegative and virus isolation negative. Application of this nested BTV PCR-based assay to clinical samples resulted in detection of BTV RNA from a variety of tissues collected from calves and deer with natural and experimental BTV infections. The described BTV PCR-based assay provides a valuable tool to study the epidemiology of BTV infection in susceptible wild ruminants and domestic livestock.     

Sensitive detection of a novel class of toluene-degrading denitrifiers, Azoarcus tolulyticus, with small-subunit rRNA primers and probes

Azoarcus tolulyticus is a new class of widely distributed toluene-degrading denitrifiers of potential importance in remediating benzene, toluene, ethylbenzene, and xylene (BTEX)-contaminated environments. To detect these organisms in the environment, 16S rRNA gene-based phylogenetic probes were developed. Two sets of specific PCR amplification primers and two oligonucleotide hybridization probes were designed and tested against both closely and distantly related environmental isolates. All of these primers and probes were specific to the species A. tolulyticus. The sensitivity of the PCR amplification primer sets was evaluated with DNA isolated from A. tolulyticus Tol-4 pure culture and from sterile soils seeded with a known number of Tol-4 and Escherichia coli cells. These primer sets were able to detect 1 fg to 1 pg of template DNA from the pure culture and 1.11 x 10(2) to 1.1 x 10(8) Tol-4 cells per g of soil in the presence of 1.56 x 10(10) E. coli cells. These two PCR amplification primers were also successfully tested at two field sites. The primers identified the A. tolulyticus strains among the toluene-degrading bacteria isolated from a low-O2-high-NO(3)- aquifer at Moffett Field, Calif. Also, the presence of A. tolulyticus was detected in the groundwater samples from a BTEX-contaminated aquifer at an industrial site in Detroit, Mich., which showed anaerobic toluene degradation.     

The effect of chemical anti-inhibitors on fibrinolytic enzymes and inhibitors

Based on published gene sequences of bovine viral diarrhoea virus (BVDV) type I and classical swine fever virus (CSFV), genus- and species-specific primers were designed to detect and identify pestivirus cDNA sequences in a nested polymerase chain reaction (PCR). The PCR primers were validated using cDNA synthesized from 146 pestivirus isolates, comprising representatives of all four so far described genotypes (BVDV type I, BVDV type II, CSFV and border disease virus), as well as others of uncertain classification. PCR products of the predicted size were amplified from all viruses with the genus-specific primers. All 53 cattle isolates, including 5 typed antigenically as BVDV type II were amplified by the internal BVDV-specific primers, but not the CSFV-specific primers. The same result was found for other BVDV type I and II viruses isolated from sheep and pigs. Seventy-seven CSF viruses were amplified by their respective internal primers. Available information strongly indicate that 4 CSF viruses also amplified by the BVDV-specific primers had been contaminated with BVDV in cell cultures. Border disease viruses were mostly not detected by the BVDV-specific primers, but were detected weakly by the CSFV-specific primer pair. Using carrier RNA for extraction of viral RNA, the sensitivity of detection of the single and nested PCR was, respectively, 5 and 50 times higher than obtained with a cell culture assay. The RT-PCR also detected BVDV in all of 15 commercial batches of fetal calf serum examined, and verified three earlier diagnoses of CSFV by detecting specific gene sequences in 30 year old frozen archival organ samples.     

Structure of Bcl-1 and IgH-CDR3 rearrangements as clonal markers in mantle cell lymphomas

Mantle cell lymphoma represent a clinicopathologically distinct entity of malignant non-Hodgkin's lymphoma (NHL) and are characterized by a specific chromosomal translocation t(11;14)(q13;q32) involving the cyclin D1 gene also designated as bcl-1/PRAD1 gene on chromosome 11 and the heavy chain immunoglobulin joining region on chromosome 14. We have established a PCR method to amplify t(11;14) junctional sequences in DNA from fresh frozen and paraffin-embedded tissue by bcl-1-specific primers in combination with a consensus immunoglobulin JH primer. A total of 65 cases histologically classified as mantle cell lymphoma (MCL) were analyzed for the presence of a t(11;14) translocation and monoclonal IgH-CDR3 rearrangements. From 26 patients with classical MCL and three cases with the anaplastic variant of MCL fresh frozen biopsy material was available for DNA extraction. We detected a bcl-1/JH rearrangement in 12 out of 29 samples (41%). In 36 cases paraffin-embedded lymph node tissue was the only source of DNA. In this material we found a bcl-1/JH rearrangement in six out of 31 samples with intact DNA (20%). To confirm the specificity of the PCR and to determine the bcl-1/JH junctional region sequences as clone-specific marker in individual patients we characterized the junctional DNA sequences by direct PCR sequencing in 16 cases. Interestingly we found that six bcl-1/JH junctions harbored DH segments in their N regions indicating that bcl-1/JH rearrangements can occur in a later stage of B cell ontogeny during which the complete VH to DH-JH joining or VH-replacement takes place. To investigate the suitability of IgH-CDR3 as sensitive molecular marker for those MCL patients in which a t(11;14) translocation can not easily be amplified, we additionally analysed 60 cases for the presence of monoclonally rearranged IgH genes by IgH-CDR3-PCR. A monoclonal IgH-CDR3 PCR product could be identified in 24 out of 29 fresh frozen samples (79%) whereas only 11 out of 31 samples (36%) with paraffin-derived DNA were positive. We demonstrate that automated fluorescence detection of monoclonal IgH-CDR3 PCR products allows the rapid and sensitive monitoring of minimal residual disease also in cases that lack a PCR amplifiable t(11;14) translocation. In combination with allele-specific primers the procedure may improve current experimental approaches for detection of occult MCL cells at initial staging and residual disease during and after therapy.     

Quantification of HIV-1 using multiple competitors in a single-tube assay

Methods for quantification of human immunodeficiency virus type 1 (HIV-1) based on competitive PCR and fragment analysis have been developed. Samples containing HIV-1 DNA and known amounts of three cloned competitors were co-amplified by PCR with semi-nested primers. The competitor DNAs contained the same long terminal repeat primer binding sequences as the wild-type DNA, but they are different in internal sequences and length. One of the inner primers was fluorescent-labeled to allow discrimination between the wild-type DNA and the three competitors by fragment analysis using a standard automated sequencer. A calibration curve using the peak area of the three competitors enabled accurate determination of target amount with minimal variations. The method presented here can be used for quantification of HIV-1 in clinical samples and will be useful for monitoring disease progression and treatment effects.     

Epidural analgesia for labour and delivery: informed consent issues

We analyzed the clade distribution of B and E in HIV-1 isolates in Japan by a nested PCR method using 5'-CCCACAAGATTTAAATATG-3' of the gag gene as clade B primer and 5'-CCCACAAGATTTAAACTCC-3' of the gag gene as clade E primer. Seventy-two anti-HIV-1 confirmatory positive serum samples were collected during a period of 1991-1996 in two hospitals in Yokohama City. Peripheral blood mononuclear cells were obtained from the buffy coat of these samples and extracted DNA were used for nested PCR. The 72 cases comprised of 11 Japanese hemophiliacs, 14 Japanese male homosexuals, 19 Japanese male heterosexuals, 5 Japanese female heterosexuals and 23 Thai female heterosexuals. Of these 36 were clade B and 35 were clade E and one case showed positive PCR results for both B and E primers. Almost all male Japanese hemophiliacs and homosexuals in our sample have clade B, while the female Thai heterosexuals have clade E, irrespective of the year of isolation. As for Japanese male heterosexuals, through 1993, clade B was predominant but since 1994, the predominate clade switched to clade E. Although the number of Japanese female heterosexuals in our sample is small, clade B was isolated in 2 cases even after 1994.     

Molecular epidemiology of methicillin-resistant Staphylococcus aureus in 12 New York hospitals. MRSA Collaborative Study Group

Hepatitis C virus (HCV) sequences recovered from serum, peripheral blood mononuclear cells (PBMCs), and various tissues from human immunodeficiency virus type 1 (HIV-1) positive patients were compared by single strand conformational polymorphism (SSCP) and sequencing. In five patients, paired serum and PBMCs samples were analyzed while in two other patients multiple autopsy tissues were studied. Sequences amplified from the NS5 and E2 regions were consistently identical in the same patient; however, three PBMCs samples and three different tissue samples (pancreas and adrenal gland in one patient and lymph node in the other patient) contained 5' untranslated region (5'UTR) sequences that were different from circulating sequences. The presence of 5' UTR sequences differing from circulating sequences correlated with the presence of HCV RNA negative strand, as the latter was detected by a Tth-based strand-specific assay in all but one of these samples. These two independent lines of evidence: viral sequence differences and the presence of RNA negative strand in the same tissues strongly argue for the genuine presence of extrahepatic HCV replication, at least in the setting of HIV-1 infection.     

PCR amplification and comparison of nucleotide sequences from the groESL heat shock operon of Ehrlichia species

Degenerate PCR primers derived from conserved regions of the eubacterial groESL heat shock operon were used to amplify groESL sequences of Ehrlichia equi, Ehrlichia phagocytophila, the agent of human granulocytic ehrlichiosis (HGE), Ehrlichia canis, Bartonella henselae, and Rickettsia rickettsii. The groESL nucleotide sequences were less conserved than the previously determined 16S rRNA gene sequences of these bacteria. A phylogenetic tree derived from deduced GroEL amino acid sequences was similar to trees based on 16S rRNA gene sequences. Nucleotide sequences obtained from clinical samples containing E. equi, E. phagocytophila, or the HGE agent were very similar (99.9 to 99.0% identity), and the deduced amino acid sequences were identical. Some divergence was evident between nucleotide sequences amplified from samples originating from the United States (E. equi and the HGE agent) and sequences from the European species, E. phagocytophila. A single pair of PCR primers derived from these sequences was used to detect E. chaffeensis and HGE agent DNA in blood samples from human patients with ehrlichiosis.     

PCR detection of North American and Central African isolates of epizootic hemorrhagic disease virus (EHDV) based on genome segment 10 of EHDV serotype 1

PCR amplification technology for the detection of epizootic hemorrhagic disease virus (EHDV) ribonucleic acid in cell culture and clinical specimens was developed. With oligoribonucleotide primers selected from genome segment 10 of EHDV serotype 1 (EHDV-1), which codes for two nonstructural proteins (NS3 and NS3a), the PCR-based assay resulted in a 535-bp PCR product. RNAs from North American EHDV-1 prototype, EHDV-2 prototype, and a number of EHDV field isolates, including the Central African isolates of EHDV-5 and EHDV-318 propagated in cell cultures, were detected by this PCR-based assay. The specific 535-bp PCR products were visualized onto agarose gels, and the identity of the PCR products was confirmed by chemiluminescent hybridization with a 352-bp internal probe. The sensitivity of the EHDV PCR assay was increased by chemiluminescent hybridization; by this EHDV-NS3 PCR, 10 fg of EHDV RNA was detected (equivalent to 600 viral particles). Amplification product was not detected when the PCR-based assay was applied to RNAs from North American bluetongue virus prototype serotypes 2, 10, 11, 13, and 17; total nucleic acid extracts from uninfected BHK-21 cells; or unfractionated blood from calves and deer that were EHDV seronegative and virus isolation negative. The described EHDV PCR-based assay with primers derived from segment 10 of EHDV-1 resulted in detection of EHDV RNA from blood and tissues collected from calves and deer with natural and experimental EHDV infections and provides a valuable tool to study the epidemiology of EHDV infection in susceptible ruminants.     

Efficient priming of PCR with short oligonucleotides conjugated to a minor groove binder

The tripeptide 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI3) binds to the minor groove of DNA with high affinity. When this minor groove binder (MGB) is conjugated to the 5'-end of short oligodeoxynucleotides (ODNs), the conjugates form unusually stable hybrids with complementary DNA in which the tethered CDPI3group resides in the minor groove. We show that these conjugates can be used as PCR primers. Due to their unusually high binding affinity, conjugates as short as 8-10mers can be used to amplify DNA with good specificity and efficiency. The reduced length primers described here might be appropriate for the PCR amplification of viral sequences which possess a high degree of variability (e.g., HPV, HIV) or for recent techniques such as gene hunting and differential display which amplify multiple sequences using short primer pairs.     

Analysis of T-DNA-mediated translational beta-glucuronidase gene fusions

Using polymerase chain reaction (PCR) with back-to-back primers, 85 different mitochondrial DNA (mtDNA) rearrangements, consisting of partial duplications or mini-circles, were detected in brain, liver, and heart tissue from Fischer 344 rats. The regions around the mitochondrial tRNALeu(UUR) gene, the cluster of three tRNA genes [His, Ser(AGY), Leu(UUC)], as well as the region of the displacement loop were analyzed separately with different primer sets. Rearrangements were detected in all regions analyzed in samples taken throughout the animal life span, ranging from 1 day old to 33 months of age (senescent). Two-thirds of the rearrangements terminated at short (3-9-bp) direct repeats. Three of the different rearrangements were detected in more than one animal; the most common rearrangement was found in nine different template preparations. Two loci (hot spots) were found to be particularly susceptible to rearrangement, and both were located at sequences that exhibited highly conserved potential for secondary structure formation. The displacement loop region of 10 samples exhibited the presence of multiple tandem duplications ranging between 324 and 449 bp in length. One of these consisted of heterologous, but overlapping, repeating units. Identical PCR protocols were carried out in control experiments using a cloned fragment of mtDNA that encompassed the most common hot spot sequence. The results showed that this fragment did not artifactually generate a rearrangement junction under our PCR conditions and suggested that this sequence does not promote rearrangement mutations in bacteria during the cloning process.     

Identification of putative sequence specific PCR primers for detection of the toxigenic fungal species Stachybotrys chartarum

The nucleotide sequence of a c 936 bp segment of the nuclear rRNA gene operon was determined for the toxigenic fungal species Stachybotrys chartarum and for other species of Stachybotrys and the related genus Memnoniella. This information was used to infer the phylogenetic relationships of these organisms and to search for sequence specific polymerase chain reaction (PCR) primers for S. chartarum in the internal transcribed spacer (ITS) regions. Searches for candidate primers were performed both by computer using the commercially available Oligo(R) v5.0 primer analysis software package and by manual inspection of the aligned sequences. Primers identified in both types of searches were evaluated for their specificities using a priming efficiency analysis algorithm available in the Oligo(R) 5.0 software. The automated computer searches were unsuccessful in finding S. chartarum-specific primers but did identify a group-specific reverse primer (designated as StacR4) for a phylogenetically related cluster of species that included S. chartarum. Manual searches led to the identification of a reverse primer (designated as StacR3) that was predicted to be specific for only S. chartarum and one other species of Stachybotrys. Experimental PCR analyses using these primers in conjunction with a universal forward primer indicated that the computer-generated amplification efficiency predictions were correct in most instances. A notable exception was the finding that StacR3 was specific only for S. chartarum. The relative merits of different PCR strategies for the detection of S. chartarum employing either one or both of the primers identified in this study are discussed.     

Use of the flagellar H7 gene as a target in multiplex PCR assays and improved specificity in identification of enterohemorrhagic Escherichia coli strains

PCR products of 1.8 kb were generated with DNAs from all Escherichia coli H7 strains tested by using oligonucleotide primers which flank the fliC gene. Three RsaI digestion profiles of these PCR products were evident on agarose gels; the first occurred with serotype O55:H7, O157:H7, or nonmotile (NM) strains, the second occurred with serotype O1:H7 and O18:H7 strains, and the third occurred with serotype O?:H7, O19:H7, O121:H7, O88:H7, and O156:H7 strains. Despite these differences, the nucleotide sequences of the E. coli E32511 (O157:NM) and U5-41 (O1:H7) fliC genes were 97% homologous. Two PCR primer pairs synthesized on the basis of the E32511 H7 fliC sequence amplified specific DNA fragments from all E. coli H7 strains, but did not amplify DNA fragments from the other bacterial strains. The H7-specific primers were used in combination with other primers which target the Verotoxin 1(VT1) and VT2 genes and the E. coli O157:H7 eaeA gene in multiplex PCR assays. In these assays, vt and eaeA PCR products were observed with DNAs from the majority of EHEC strains and vt, eaeA, and fliC PCR products were observed with DNAs from E. coli O157:H7 or NM strains. Only eaeA PCR products were present with DNA from enteropathogenic E. coli, and only vt PCR products occurred with VT-producing E. coli which are not EHEC. The multiplex PCR assays described allow for the specific identification of E. coli O157:H7 or NM and other EHEC strains.     

Introduction to the special section on attachment and psychopathology: 2. Overview of the field of attachment

An improved system for amplification of hepatitis C virus genome (HCV) was developed based on a multiplex nested polymerase chain reaction format. Two sets of oligonucleotide primers were used simultaneously. One was derived from the conserved sequences in the 5' non-coding region of the viral genome which can bind to the viral genome of all genotypes. The other set of primers was designed from a sequence in the nonstructural-5 region of HCV. HCV genotypes 1 and 3 can be differentiated by the banding patterns of amplified DNA products. All of 39 samples containing the HCV genotype 1 could be amplified with primers in the 5' non-coding region only, whereas 92% of those with genotype 3 could be amplified by both primer sets. In addition, HCV RNA can be detected in 81% of 84 anti-HCV-positive blood donors and in 0% of 34 anti-HCV-negative cases. Of the HCV RNA-positive specimens, 69% showed genotype 1-like patterns while 31% showed genotype 3-like patterns. The detection rate of HCV RNA in this study was much higher than that in our previous report due to the improvement of new primers which can detect all genotypes of the virus. In conclusion, this improved amplification system is a sensitive method for rapid identification of HCV RNA in clinical specimens that can simultaneously differentiate the two most common genotypes of HCV found in Thailand.     

Detection of soluble methane monooxygenase producing Methylosinus trichosporium OB3b by polymerase chain reaction

The soluble methane monooxygenase (sMMO) enzyme complex of methanotrophs cometabolizes haloaliphatic compounds such as trichloroethylene. Two 18-mer oligonucleotides as primary primers and a nested primer of the same length were selected to amplify specific DNA sequences of the sMMO gene cluster using polymerase chain reaction (PCR). Two DNA fragments of sizes 270 and 400 base pairs were obtained when purified DNA from the methanotroph Methylosinus trichosporium OB3b was used as template. The primers were specific for sMMO sequences of M. trichosporium, since none of the 13 bacterial isolates screened yielded the expected length of PCR-amplified DNA fragments. The detection limit of the PCR method was 5 x 10(2) cells of M. trichosporium. The sMMO sequences were successfully amplified in groundwater (containing native microbial population) when seeded with M. trichosporium, FP1 sense (5'-ATGTCCAGCGCTCATAAC-3'), RP1 antisense (5'-TCAGATGTCGGTCAGGGC-3'), FP2 sense nested (5'GCCATCATCGGTCAGGGC-3'), and FP2 sense nested (5'-GCCATCATCGAGGACATC-3').