PCR-based Reverse Line Blot Research Articles

A multiplex PCR‐based reverse line blot hybridization (mPCR/RLB) assay for detection of bacterial respiratory pathogens in children with pneumonia

To develop and evaluate a novel method for simultaneous identification of 12 potential bacterial pathogens in children with community-acquired pneumonia. A multiplex PCR-based reverse line blot (mPCR/RLB) assay was developed, to identify 12 respiratory bacterial pathogens, namely Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Moraxella catarrhalis, Haemophilus influenzae, Haemophilus influenzae type b, Bordetella pertussis, Klebsiella pneumoniae, Legionella pneumophila, Mycobacterium tuberculosis, Chlamydia pneumoniae, Mycoplasma pneumoniae, and single (uniplex) PCRs were used for S. pneumoniae and H. influenzae only. In a preliminary evaluation, we compared the results of mPCR/RLB with those of single (uniplex) PCRs and culture of nasopharyngeal aspirates (NPAs) from 100 children under 5 years, admitted to Beijing Children's Hospital between October 2004 and May 2005, with pneumonia. Reference strains and clinical isolates of all 12 target species were correctly identified by mPCR/RLB. Potential pathogens were isolated from one blood culture and 26% of respiratory cultures. One or more pathogens were identified in 70% of respiratory specimens--by mPCR/RLB in 63%, uPCR only in another 3%, culture only in 2%, and culture plus uPCR in 2%. The species most commonly identified were S. pneumoniae (54%) and H. influenzae (38%, including type b, 4%). Cultures were not performed for B. pertussis, M. tuberculosis, C. pneumoniae or M. pneumoniae but each was identified by mPCR/RLB in between one and four specimens. Two or more potential pathogens were identified in 35% of specimens. Ten of 14 S. pneumoniae isolates belonged to serotypes represented in the 11-valent pneumococcal conjugate vaccine. The mPCR/RLB assay is a sensitive tool for identification of respiratory pathogens, including mixed infections and bacteria requiring special culture methods.

Phenotypical and genotypical characteristics of invasive group B Streptococcus isolates in Western Sydney 2000-2005

To analyse antimicrobial susceptibility and serotypes of group B streptococcus (GBS) bloodstream isolates from different patient groups. Susceptibility to penicillin, erythromycin and clindamycin was measured for 99 bloodstream GBS isolates collected between October 2000 and July 2005. Multiplex PCR-based reverse line blot (mPCR/RLB) assays were used to identify macrolide resistance genes and capsular serotype for each isolate. Clinical correlation was obtained from chart review. Adult bacteraemia accounted for 84 of 99 (85%) isolates, and were usually associated with underlying diseases such as diabetes, malignancy and renal failure. Overall mortality was 10%. Known macrolide resistance genes [ermB (2), ermA/TR (3) and mefA/E (2)] were detected in seven of eight erythromycin resistance isolates. Four of these isolates expressed MLSB phenotype, two with constitutive (ermB) and two with inducible (ermA/TR) clindamycin resistance. Of four M phenotype isolates, two had mefA/E, one had ermA/TR and one had no detectable macrolide resistance genes. Serotype III was significantly more common in neonatal isolates; serotype V was more common among adult isolates and was associated with increased mortality. mPCR/RLB is a rapid molecular method to identify GBS serotype and macrolide resistance genes. This is the first major study correlating these characteristics with demographic data for invasive isolates.

Identification of Less-Common Streptococcus pneumoniae Serotypes by a Multiplex PCR-Based Reverse Line Blot Hybridization Assay

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.

Three New Macrolide Efflux ( mef ) Gene Variants in Streptococcus agalactiae

Streptococcus agalactiae (group B streptococcus [GBS]) is the most common cause of neonatal sepsis (9). Penicillin is used for intrapartum prophylaxis, but erythromycin or clindamycin is recommended for patients allergic to penicillin (3). There are two major mechanisms of erythromycin resistance in S. agalactiae, (i) erythromycin ribosomal methylase, mediated by ermB, ermA, ermTR, or ermC, which confers cross-resistance to macrolides, lincosamides, and streptogramin B (MLSB phenotype), and (ii) a less common macrolide efflux pump, mediated by mef (7), which confers resistance to 14- and 15-membered macrolides only (M phenotype). The major mef variants, mefE and mefA, were originally identified in S. pneumoniae and S. pyogenes, respectively (5, 11); both are found in S. agalactiae (2), although mefE is much more common (4, 12). Recently, we tested 512 GBS isolates from Australia, Hong Kong, and South Korea by using a multiplex PCR-based reverse line blot (mPCR/RLB) assay to identify nine resistance markers and identified mef in 22 (12). However, we did not distinguish mef variants. Subsequently, we tested a total of 1,629 GBS clinical isolates (including the original 512) from nine countries by using the same mPCR/RLB, except that two new probe pairs, specific for mefA and mefE, were added (Table ​(Table1).1). Isolates were typed by using a three-set genotyping system which identifies the molecular serotype (MS), surface protein genes, and mobile genetic elements, as described previously (8). Antibiotic susceptibilities to erythromycin, clindamycin, and tetracycline were measured by E-test (AB Biodisk; Australia Laboratory Services Pty. Ltd.) and interpreted as recommended by the Clinical and Laboratory Standards Institute (12). TABLE 1. Oligonucleotide primers and probes used in this study Forty five (2.7%) of 1,629 isolates were positive for mef, and of these, 35 contained mefE, 7 contained mefA, and 3 gave weak or variable signals with mefE- and mefA-specific probes. These three isolates were among 16 mef-positive isolates from Hong Kong. Their genotypes and phenotypic susceptibilities to erythromycin, clindamycin, and tetracycline are shown in Table ​Table2.2. All three had the M phenotype and MS Ia but atypical genotypes. MS Ia usually has the surface protein gene alp1 and insertion sequence IS1381 (8, 10). Two of these isolates had alp1 but, instead of IS1381, carried the type II intron GBSi1, usually found in MS III but rarely in other serotypes (10). The other isolate had neither the surface protein gene nor the insertion sequence. TABLE 2. Characteristics and comprehensive genotyping results of three mef variants From each of these three isolates, mef was amplified and sequenced with the primers shown in Table ​Table1.1. The full sequences indicated that all were novel mef variants not previously described in a GBS. They were deposited in GenBank with accession numbers {type:entrez-nucleotide-range,attrs:{text:DQ445269 to DQ445271,start_term:DQ445269,end_term:DQ445271,start_term_id:90902659,end_term_id:90902663}}DQ445269 to DQ445271. {type:entrez-nucleotide,attrs:{text:DQ445271,term_id:90902663,term_text:DQ445271}}DQ445271 and {type:entrez-nucleotide,attrs:{text:DQ445270,term_id:90902661,term_text:DQ445270}}DQ445270 were 99% similar to each other but only 88% and 89% homologous with mefE (GenBank accession no. {type:entrez-nucleotide,attrs:{text:AF227521,term_id:18478326,term_text:AF227521}}AF227521) and mefA (GenBank accession no. {type:entrez-nucleotide,attrs:{text:AY064721,term_id:22121176,term_text:AY064721}}AY064721), respectively. They shared 99 to 100% homology with a mef variant recently identified in Streptococcus dysgalactiae (a group G streptococcus) (GenBank accession no. {type:entrez-nucleotide,attrs:{text:AM168138,term_id:83582439,term_text:AM168138}}AM168138 and {type:entrez-nucleotide,attrs:{text:AY355405,term_id:34147931,term_text:AY355405}}AY355405) (1). {type:entrez-nucleotide,attrs:{text:DQ445269,term_id:90902659,term_text:DQ445269}}DQ445269 has not been described before; it had 89% homology with mefA (GenBank accession no. {type:entrez-nucleotide,attrs:{text:AY064721,term_id:22121176,term_text:AY064721}}AY064721) and the novel group G streptococcus mef gene (GenBank accession no. {type:entrez-nucleotide,attrs:{text:AY355405,term_id:34147931,term_text:AY355405}}AY355405), 91% homology with mefE (GenBank accession no. {type:entrez-nucleotide,attrs:{text:AY227521,term_id:29653143,term_text:AY227521}}AY227521), and 92% homology with another mef variant, mefI, described in Streptococcus pneumoniae (GenBank accession no. {type:entrez-nucleotide,attrs:{text:AJ971089,term_id:194032949,term_text:AJ971089}}AJ971089) (6). The inconsistent mPCR/RLB results for these isolates can be explained by mutations in the mefAESb and mefAEAb regions. New primers and probes will be required to detect them reliably by mPCR/RLB. For these novel mef variants, we propose the names mefG (for {type:entrez-nucleotide,attrs:{text:DQ445270,term_id:90902661,term_text:DQ445270}}DQ445270 and {type:entrez-nucleotide,attrs:{text:DQ445271,term_id:90902663,term_text:DQ445271}}DQ445271) and mefB (for {type:entrez-nucleotide,attrs:{text:DQ445269,term_id:90902659,term_text:DQ445269}}DQ445269) to reflect the beta-hemolytic streptococcus groups in which they were first identified. These findings and the atypical genotype patterns suggest that these strains have arisen by recombination. Further investigation will be required to determine their clinical significance.

Multiplex PCR-based reverse line blot hybridization assay (mPCR/RLB)—a practical epidemiological and diagnostic tool

Combining multiplex PCR, sequentially, with reverse line blot hybridization (mPCR/RLB) is a convenient, objective way to identify up to 43 targets in 43 individual specimens simultaneously (using a 45-lane membrane format). It is more flexible and less expensive than DNA microarray. The number of targets is adequate for epidemiological and most clinical diagnostic applications; based on the same target (43) and specimen numbers (43), it is much more practical than conventional uniplex PCR (uPCR) and mPCR. We have used the protocol to identify and subtype bacteria, viruses and fungi and identify pathogens in clinical specimens; potentially, it could be used for many other applications, such as detection of mutations in, or identification of alleles of, eukaryotic genes. Development of each assay involves (i) careful primer and probe design, based on literature and sequence database searches, which are critical to success of the assay; and (ii) bench-top evaluation, using known samples, controls and dilution series, to confirm sensitivity, specificity and reproducibility. The assay takes about one and half working days to complete; about 4 h for the mPCR and 6 h for the RLB, including a total of 4 h 'hands-on' time.

Evaluation of a Multiplex PCR-Based Reverse Line Blot-Hybridization Assay for Identification of Serotype and Surface Protein Antigens of Streptococcus agalactiae

A 33-primer multiplex PCR-based reverse line blot (mPCR/RLB) assay was developed to identify Streptococcus agalactiae serotypes and surface protein antigen genes simultaneously. It was evaluated by using 551 clinical isolates. The mPCR/RLB assay was more sensitive than conventional serotyping, especially for protein antigen typing, but otherwise the results correlated well.

Molecular serotype identification ofStreptococcus agalactiaeof bovine origin by multiplex PCR-based reverse line blot (mPCR/RLB) hybridization assay

We used a multiplex PCR-based reverse line blot (mPCR/RLB) hybridization assay and sequencing of a variable region of the cps cluster to identify serotypes of 140 Streptococcus agalactiae (group B Streptococcus; GBS) isolates from cattle. Only 71 (51%) isolates were typeable using antisera, but molecular serotypes (MS) were assigned to 133 (95%) and 139 (99%) isolates by partial cpsE-cpsF-cpsG sequencing and mPCR/RLB, respectively. Ninety-four isolates (67%) belonged to MS III and most belonged to a molecular serosubtype (msst) III-3, which is uncommon among GBS isolates from humans. Our results demonstrate that cps clusters of bovine GBS differ significantly from those of GBS isolates from humans.

Multiplex PCR-Based Reverse Line Blot Hybridization Assay To Identify 23 Streptococcus pneumoniae Polysaccharide Vaccine Serotypes

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.

Reverse Line Blot Assay for Direct Identification of Seven Streptococcus agalactiae Major Surface Protein Antigen Genes

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.

Simultaneous Detection of Nine Antibiotic Resistance-Related Genes in Streptococcus agalactiae Using Multiplex PCR and Reverse Line Blot Hybridization Assay

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.

Is the perceived association between Chlamydia pneumoniae and vascular diseases biased by methodology?

Inter- and intralaboratory inconsistencies in detection rates of Chlamydia pneumoniae in vascular specimens have been demonstrated. In this study, 66 vascular tissue specimens from 66 patients with vascular disease were tested by three PCR assays: a 16S PCR-based reverse line blot (RLB) assay, a single-step PCR, and a nested PCR. Also, we explored the impacts of different DNA polymerase enzymes on the results based on gel electrophoresis and hybridization. The PCR results by gel electrophoresis in the single-step PCR depended on which DNA polymerase was used. All samples were negative with AmpliTaq Gold DNA polymerase, and 54.5% (36 of 66) were positive with the conventional Taq DNA polymerase. All samples were negative after hybridization with a C. pneumoniae-specific probe. In the nested PCR, all specimens were negative by gel electrophoresis and after hybridization. The RLB assay failed to detect C. pneumoniae in any specimen; however, 20 specimens were Chlamydia sp. positive. The sequence analysis of six of these samples demonstrated Chlamydia-like organisms. RLB detected Chlamydia sp. DNA in water and in the elution buffer after passage of the Qiagen columns (11 of 40). This study identified factors that may influence the detection of C. pneumoniae DNA in vascular tissues and consequently bias the perception of a link between C. pneumoniae and vascular diseases. The following are strongly recommended: to use DNA polymerases that have to be activated, to decontaminate with dUTP-uracil-DNA glycosylase, to hybridize with specific probes, to include sufficient controls, and to use molecular grade water.

Reverse line blot hybridization used to identify hemoprotozoa in Minorcan cattle.

Piroplasmosis, a tick-borne protozoal disease, is an important disease affecting domestic and wild animals. We performed PCR-based reverse line blot hybridization (RLB) assays on blood samples obtained from 133 cattle exposed to ticks in field conditions in Minorca (Balearic Islands, Spain) in three different seasons. The oligonucleotides used were those for Theileria annulata, T. buffeli, T. taurotragi, T. velifera, Babesia bigemina, B. bovis, B. divergens, and B. major. The RLB technique allowed the simultaneous identification of T. annulata, T. buffeli, B. bigemina, and B. bovis as the piroplasms present in cattle in Minorca. Of the 133 animals, only 4 were not infected by any of the studied parasites. The results indicated endemic piroplasm infection in cattle in Minorca; especially important was the presence of T. annulata. The RLB was highly sensitive and allowed the simultaneous detection and identification of the Theileria and Babesia species in carrier cattle, which cannot be achieved by classical identification methods.

7.4#142 - PCR-based reverse line blot typing system for the HLA-DQB1 locus

7.4#142 - PCR-based reverse line blot typing system for the HLA-DQB1 locus