Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Reverse line blot assay for direct identification of seven Streptococcus agalactiae major surface protein antigen genes.

ABSTRACT: We developed a multiplex PCR-based reverse line blot hybridization assay (mPCR/RLB) to detect the genes encoding members of the family of variable surface-localized proteins of Streptococcus agalactiae (group B streptococcus [GBS]), namely, Bca (Calpha), Rib, Epsilon (Epsilon/Alp1/Alp5), Alp2, Alp3, and Alp4, and the immunoglobulin A binding protein, Bac (Cbeta). We used the assay to identify these genes in a collection of well-characterized GBS isolates and reference strains. The results showed that mPCR/RLB avoids the common problems of cross-reaction and nontypability associated with protein typing using antisera. It is as sensitive as, but more practical than, separate gene-specific PCRs and would be suitable for large molecular epidemiological studies of GBS.

SUBMITTER: Zhao Z

PROVIDER: S-EPMC1356620 | biostudies-literature | 2006 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Reverse line blot assay for direct identification of seven Streptococcus agalactiae major surface protein antigen genes.

Zhao Zuotao Z Kong Fanrong F Gilbert Gwendolyn L GL

Clinical and vaccine immunology : CVI 20060101 1

We developed a multiplex PCR-based reverse line blot hybridization assay (mPCR/RLB) to detect the genes encoding members of the family of variable surface-localized proteins of Streptococcus agalactiae (group B streptococcus [GBS]), namely, Bca (Calpha), Rib, Epsilon (Epsilon/Alp1/Alp5), Alp2, Alp3, and Alp4, and the immunoglobulin A binding protein, Bac (Cbeta). We used the assay to identify these genes in a collection of well-characterized GBS isolates and reference strains. The results showed ...[more]

PMID: 16426012

Similar Datasets

Simultaneous detection of nine antibiotic resistance-related genes in Streptococcus agalactiae using multiplex PCR and reverse line blot hybridization assay.

Project description:Streptococcus agalactiae (group B streptococcus [GBS]) is the leading cause of neonatal and maternal sepsis. Penicillin is recommended for intrapartum prophylaxis, but erythromycin or clindamycin is used for penicillin-allergic carriers. Antibiotic resistance (AR) has increased recently and needs to be monitored. We have developed a multiplex PCR-based reverse line blot (mPCR/RLB) hybridization assay to detect, simultaneously, seven genes encoding AR--erm(A/TR), erm(B), mef(A/E), tet(M), tet(O), aphA-3, and aad-6--and two AR-related genes, int-Tn and mreA. We tested 512 GBS isolates from Asia and Australasia and compared mPCR/RLB with antibiotic susceptibility phenotype or single-gene PCR. Phenotypic resistance to tetracycline was identified in 450 (88%) isolates, of which 442 had tet(M) (93%) and/or tet(O) (6%). Of 67 (13%) erythromycin-resistant isolates, 18 were susceptible to clindamycin, i.e., had the M phenotype, encoded by mef(A/E); 39 had constitutive (cMLS(B)) and 10 inducible clindamycin resistance, and of these, 34 contained erm(B) and 12 erm(A/TR). Of four additional isolates with mef(A/E), three contained erm(B) with cMLS(B) and one was erythromycin susceptible. Of 61 (12%) clindamycin-resistant isolates, 20 were susceptible to erythromycin and two had intermediate resistance. Based on sequencing, 21 of 22 isolates with mef had mef(E), and 8 of 353 with int-Tn had an atypical sequence. Several AR genes, erm(B), tet(O), aphA-3, aad-6, and mef(A/E), were significantly more common among Asian than Australasian isolates, and there were significant differences in distribution of AR genes between GBS serotypes. Our mPCR/RLB assay is simple, rapid, and suitable for surveillance of antibiotic resistance in GBS.

| S-EPMC1346803 | biostudies-literature

Multiplex PCR-based reverse line blot hybridization assay to identify 23 Streptococcus pneumoniae polysaccharide vaccine serotypes.

Project description:We developed a multiplex PCR-based reverse line blot assay to identify 23 pneumococcal serotypes represented in the polysaccharide vaccine, using 334 well-characterized isolates, representing all 90 serotypes, and 268 "unknowns." The assay identified all target serotypes, but 11, which cross-react with 1 to 4 nonvaccine serotypes, could be distinguished using serotype-specific antisera.

| S-EPMC1479170 | biostudies-literature

Identification of less-common Streptococcus pneumoniae serotypes by a multiplex PCR-based reverse line blot hybridization assay.

Project description:We developed a multiplex PCR-based reverse line blot (mPCR/RLB) assay to identify 50 uncommon pneumococcal serotypes. In combination with a previously described mPCR/RLB assay (3), all 90 pneumococcal serotypes can be identified individually (32 serotypes) or, because of predictable cross-reactions, to within small groups of two to five related serotypes (58 serotypes), which can be distinguished using serotype-specific antisera.

| S-EPMC2045372 | biostudies-literature

Reverse line blot hybridization assay for identification of medically important fungi from culture and clinical specimens.

Project description:We evaluated a combined panfungal PCR-reverse line blot (RLB) hybridization assay based on internal transcribed spacer 1 (ITS1) and ITS2 region polymorphisms to identify 159 Candida, Cryptococcus neoformans, and Aspergillus isolates (22 species). Its utility to identify fungal pathogens directly from 27 clinical specimens was also determined. ITS sequence analysis was performed to resolve discrepant identifications or where no RLB result was obtained. Species-specific ITS2- and ITS1-based probes correctly identified 155 of 159 isolates (98%) and 149 (93.7%) isolates, respectively. All strains were unambiguously differentiated with the exception of cross-reactivity between the Candida norvegensis probe and Candida haemulonii DNA product. Species identification of the pathogen was made for all 21 specimens (sensitivity of 100%) where species-specific probes were included in the RLB; however, there was no ITS2 probe-based hybridization signal for two specimens. Results were concordant with the culture results for 18 (85.7%) specimens. The assay was able to provide species identification in the absence of a culture result (two specimens) and to detect mixed infection (one specimen). The results indicate that the RLB assay is capable of reliably detecting yeasts and Aspergillus spp. in clinical specimens and that the incorporation of both ITS1- and ITS2-targeted probes is required for optimal sensitivity. The test has potential utility in the early diagnosis of invasive fungal infection, since "fungal" DNA was detected in all 27 specimens. Prior to incorporation of probes to detect other fungal species, ITS sequencing may be performed to achieve species identification.

| S-EPMC2045312 | biostudies-literature

Identification of 20 common human enterovirus serotypes by use of a reverse transcription-PCR-based reverse line blot hybridization assay.

Project description:The more than 100 human enterovirus (HEV) serotypes can also be classified into four species, HEV-A to -D, based on phylogenetic analysis of multiple gene regions. Current molecular typing methods depend largely on reverse transcription-PCR (RT-PCR) amplification and nucleotide sequencing of the entire or 3' half of the VP1 gene. An RT-PCR-based reverse line blot (RLB) hybridization assay was developed as a rapid and efficient approach to characterize common HEVs. Twenty HEV serotypes accounted for 87.1% of all HEVs isolated at an Australian reference virology laboratory from 1979 to 2007. VP1 sequences of all known HEV prototype strains were aligned to design one sense primer and three antisense primers for RT-PCR. After sequencing of the complete VP1 genes of 37 previously serotyped examples of the commonest 20 serotypes and alignment of these VP1 sequences with GenBank sequences, four serotype-specific probes for each serotype were designed for RLB. The RT-PCR-RLB assay was then applied to 132 HEV isolates, made up of the previously sequenced 37 isolates and another 95 serotyped clinical isolates. The RT-PCR-RLB genotypes corresponded with the serotypes for 131/132 isolates; the one exception was confirmed by VP1 sequencing, and the genotype was confirmed by repeat conventional serotyping. Genotyping by RT-PCR-RLB complements traditional serotyping methods and VP1 sequencing and has the advantages of convenience, speed, and accuracy. RT-PCR-RLB allows detection of specific enteroviral serotypes or genotypes associated with HEV outbreaks and significant disease.

| S-EPMC2738090 | biostudies-literature

Investigating genome reduction of Bordetella pertussis using a multiplex PCR-based reverse line blot assay (mPCR/RLB).

Project description:BACKGROUND: The genetic composition of the bacterium causing whooping cough, Bordetella pertussis, has been investigated using microarray studies in order to examine potential genetic contributors to the disease re-emergence in the past decade. Regions of difference (RDs) have been previously identified as clusters of genes flanked by insertion sequences which are variably present in different sets of isolates, and have also been shown to be potential markers of B. pertussis evolution.This study used microarray data to identify and select a panel of RDs; primers and probes for these RDs were then designed to test for the presence or absence of these regions in a novel and less expensive multiplex PCR-based reverse line blot (mPCR/RLB) assay. By comparing the presence or absence of RDs, we aimed to determine the genomic variability of a diverse collection of B. pertussis strains and how they have changed over time. RESULTS: A B. pertussis specific mPCR/RLB using 43 genes representing 30 RDs, was developed and used to characterise a set of 42 B. pertussis isolates. When mapped against the previously identified evolutionary relationships of the strains, the losses of two RDs--BP0910A--BP00930 and BP1948-BP1962--were found to be associated with significant events in B. pertussis history: the loss of BP0910A--BP00930 coincided with introduction of whole cell vaccines in the 1950s while that of BP1948-BP1962 occurred after the introduction of acellular vaccines. The loss of BP1948-BP1962 also coincided with expansion of the most recent B. pertussis strains. CONCLUSIONS: The mPCR/RLB assay offers an inexpensive and fast method of determining the gene content of B. pertussis strains and also confirms that gene losses are an ongoing feature of B. pertussis evolution.

| S-EPMC4209057 | biostudies-literature

Rapid diagnosis of extensively drug-resistant tuberculosis by use of a reverse line blot hybridization assay.

Project description:Drug resistance in tuberculosis (TB) is a matter of grave concern for TB control programs, as there is currently no cure for some extensively drug-resistant (XDR) strains. There is concern that this resistance could transmit, stressing the need for additional control measures, rapid diagnostic methods, and newer drugs for treatment. We developed an in-house assay that can rapidly detect resistance to drugs involved in the definition of XDR-TB directly from smear-positive specimens. Two hundred fifteen phenotypically XDR-TB isolates and 50 pansusceptible isolates were analyzed using a reverse line blot hybridization (RLBH) assay. The assay was also successfully applied to 73 smear-positive clinical specimens. The RLBH assay exhibited good sensitivity for the detection of resistance to isoniazid (99%), rifampin (99%), fluoroquinolones (95.3%), and second-line aminoglycosides (94.8%). The results from application of this assay on direct smear-positive clinical specimens revealed 93% concordance with the phenotypic drug susceptibility test (DST) results for the above-mentioned drugs. The time to accurate DST results was significantly reduced from weeks to 3 days. This molecular assay is a highly accurate tool for screening for XDR-TB, which achieves a substantial reduction in diagnostic delays.

| S-EPMC3147869 | biostudies-other

Multiplex PCR-based reverse line blot assay for simultaneous detection of 22 virulence genes in uropathogenic Escherichia coli.

Project description:Urinary tract infections (UTIs) are among the most common bacterial infections and are responsible for significant morbidity and health care costs worldwide. The main bacterial cause of uncomplicated UTI is Escherichia coli, which possesses numerous virulence factors (VFs). Many studies of the pathogenesis of E. coli UTI have centered on VF genes. Hence, the development of better molecular assays to study VF genes would facilitate these studies. We developed a highly sensitive and specific multiplex PCR-based reverse line blot (mPCR/RLB) assay to simultaneously detect 22 VF genes of uropathogenic E. coli and then used it to characterize 180 isolates from nonpregnant women of child-bearing age with cystitis and 153 fecal isolates from similar-age healthy women, in regional New South Wales, Australia. The assay accurately identified all VF genes (of the 22 under study) known to be present in 30 previously characterized control strains. The detection limits were 28 ng of DNA from E. coli isolates and 50 CFU/ml in mock-infected urine specimens containing known concentrations of E. coli. Cystitis isolates (compared to the fecal isolates) showed a significantly higher prevalence of 18 individual VF genes and contained significantly more VF genes per isolate (median number, 18.5 versus 6.5 [P = 0.001]). Discordance between paired probes for a given VF gene occurred in several clinical test isolates but no reference strains and among the test isolates was associated with fecal source (10% of VF genes versus 2% for cystitis isolates [P < 0.001]). This novel mPCR/RLB method is a potentially powerful tool for investigating the prevalence and distribution of VFs in E. coli.

| S-EPMC3272995 | biostudies-literature

Molecular diagnosis of Old World cutaneous leishmaniasis and species identification by use of a reverse line blot hybridization assay.

Project description:Reverse line blot hybridization assays (RLB) have been used for the rapid diagnosis and genotyping of many pathogens. The leishmaniases are caused by a large number of species, and rapid, accurate parasite characterization is important in deciding on appropriate therapy. Fourteen oligonucleotide probes, 2 genus specific and 12 species specific (2 specific for Leishmania major, 3 for L. tropica, 1 for L. infantum, 3 for L. donovani, and 3 for L. aethiopica), were prepared by using DNA sequences in the internal transcribed spacer 1 (ITS1) region of the rRNA genes. Probe specificity was evaluated by amplifying DNA from 21 reference strains using biotinylated ITS1 PCR primers and the RLB. The genus-specific probes, PP and PP3', recognized all Leishmania species examined, while the species-specific probes were able to distinguish between all the Old World Leishmania species. Titrations using purified parasite DNA showed that the RLB is 10- to 100-fold more sensitive than ITS1 PCR and can detect <0.1 pg DNA. The RLB was compared to kinetoplast DNA (kDNA) and ITS1 PCR by using 67 samples from suspected cutaneous leishmaniasis (CL) patients in Israel and the West Bank. The RLB accurately identified 58/59 confirmed positive samples as CL, a result similar to that found by kDNA PCR (59/59) and better than that by ITS1 PCR (50/59). The positive predictive value and negative predictive value of the RLB were 95.1% and 83.3%, respectively. L. major or L. tropica was identified by the RLB in 55 of the confirmed positive cases, a level of accuracy better than that of ITS1 PCR with restriction fragment length polymorphism (42/59). Thus, RLB can be used to diagnose and characterize Old World CL.

| S-EPMC2546705 | biostudies-literature

Use of PCR and reverse line blot hybridization assay for rapid simultaneous detection and serovar identification of Chlamydia trachomatis.

Project description:The aim of this study was to develop and evaluate multiplex and nested PCR-reverse line blot (RLB) hybridization assays for detection and serovar identification of Chlamydia trachomatis. Two sets of primers targeting the VD2 region of the omp1 gene and one set targeting the cryptic plasmid were designed for use in multiplex (both targets) and nested PCR (omp1 only). For the RLB assay, labeled omp1 amplicons were hybridized to a membrane containing probes specific for 15 C. trachomatis serovars. The assays were used to test 429 clinical specimens, which had been previously tested for C. trachomatis using the COBAS AMPLICOR system. Specimens were tested without knowledge of the COBAS AMPLICOR result. Of 205 specimens that were positive by COBAS AMPLICOR, 201 (98%) were positive by multiplex PCR-RLB and 188 (92%) were also positive by omp1 nested PCR-RLB. In addition, three of 224 COBAS AMPLICOR-negative specimens were positive by omp1 nested PCR-RLB. One hundred sixty-six of 191 (87%) specimens in which C. trachomatis serovars were identified contained only one serovar and 25 (13%) contained two or three serovars. Serovars D, E, and F were found in 31 (16%), 83 (43%), and 51 (27%) specimens, respectively. Serovar E (41%) was the most commonly identified single serovar. Serovars J and K were found alone uncommonly (<2% each), but 18 of 25 (72%) specimens with multiple C. trachomatis serovars contained one or both (10 specimens) of these serovars. The nested (ompI) PCR-RLB is a specific and sensitive method for simultaneous detection and serovar identification of C. trachomatis, which can reliably identify mixed C. trachomatis serovars. It is suitable for use in epidemiological studies.

| S-EPMC1448689 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data