scholarly journals Multiplex PCR and Reverse Line Blot Hybridization Assay (mPCR/RLB)

10.3791/2781 ◽  
2011 ◽  
Author(s):  
Matthew V. N. O'Sullivan ◽  
Fei Zhou ◽  
Vitali Sintchenko ◽  
Fanrong Kong ◽  
Gwendolyn L. Gilbert
Keyword(s):  
Multiplex Pcr ◽  
Blot Hybridization ◽  
Reverse Line Blot ◽  
Hybridization Assay

scholarly journals A new multiplex PCR-based reverse line-blot hybridization (mPCR/RLB) assay for rapid staphylococcal cassette chromosome mec (SCCmec) typing

2009 ◽  
Vol 58 (8) ◽  
pp. 1045-1057 ◽  
Author(s):  
Lin Cai ◽  
Fanrong Kong ◽  
Qinning Wang ◽  
Huiping Wang ◽  
Meng Xiao ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Clinical Laboratory ◽  
Blot Hybridization ◽  
Rapid Identification ◽  
Reverse Line Blot ◽  
Discriminatory Power ◽  
Hybridization Assay ◽  
Coagulase Negative Staphylococci ◽  
Primer Sets ◽  
Mrsa Strains

The aim of this study was to develop a new discriminatory method for MRSA SCCmec typing based on multiplex PCR-based reverse line-blot hybridization (mPCR/RLB) assay to enable rapid identification and classification of MRSA SCCmec types in a clinical laboratory. Forty-five primer sets and 49 probes were designed and tested in uniplex PCR (uPCR) and mPCR/RLB. Probes were compared in silico to 14 whole-genome sequences and 18 partial SCCmec gene sequences of Staphylococcus aureus and complete genome and partial SCCmec genes of seven non-MRSA strains, including meticillin-susceptible S. aureus and meticillin-resistant coagulase-negative staphylococci. The method was tested on a set of 42 well-characterized reference MRSA strains. It identified all five epidemiologically relevant SCCmec types and 26 subtypes, including established and new subtypes of SCCmec III, IV (eight subtypes each) and V (three subtypes). The discriminatory power of mPCR/RLB SCCmec typing was similar to that of MLST and spa typing (Simpson indices of diversity of 0.916, 0.926 and 0.882, respectively; differences not statistically significant). The application of mPCR/RLB hybridization assay to MRSA SCCmec typing can improve the specificity, discriminatory power and throughput of the typing procedure. The detection of up to 43 mPCR products in a single hybridization assay transforms MRSA SCCmec typing from passive epidemiological library typing into a potential tool for near-real-time infection control surveillance and tracking of MRSA transmission in hospitals.

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

2006 ◽  
Vol 50 (1) ◽  
pp. 204-209 ◽  
Author(s):  
Xianyu Zeng ◽  
Fanrong Kong ◽  
Hui Wang ◽  
Archie Darbar ◽  
Gwendolyn L. Gilbert
Keyword(s):  
Antibiotic Resistance ◽  
Multiplex Pcr ◽  
Streptococcus Agalactiae ◽  
Single Gene ◽  
Blot Hybridization ◽  
Simultaneous Detection ◽  
Reverse Line Blot ◽  
Hybridization Assay ◽  
Inducible Clindamycin Resistance ◽  
Phenotypic Resistance

ABSTRACT 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 (cMLSB) 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 cMLSB 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.

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

2006 ◽  
Vol 263 (2) ◽  
pp. 236-239 ◽  
Author(s):  
Zuotao Zhao ◽  
Fanrong Kong ◽  
Gabriela Martinez ◽  
Xianyu Zeng ◽  
Marcelo Gottschalk ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Reverse Line Blot ◽  
Hybridization Assay ◽  
Serotype Identification

Epidemiology of Babesia, Anaplasma and Trypanosoma species using a new expanded reverse line blot hybridization assay

2018 ◽  
Vol 9 (2) ◽  
pp. 155-163 ◽  
Author(s):  
Martina Soledad Paoletta ◽  
Ludmila López Arias ◽  
Sofía de la Fournière ◽  
Eliana Carolina Guillemi ◽  
Carlos Luciani ◽  
...  
Keyword(s):  
Blot Hybridization ◽  
Reverse Line Blot ◽  
Hybridization Assay ◽  
Trypanosoma Species
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