PCR–Restriction Fragment Length Polymorphism and Pulsed-Field Gel Electrophoresis Characterization of Listeria monocytogenes Isolates from Ready-to-Eat Foods, the Food Processing Environment, and Clinical Samples in South Africa

2020 ◽  
Vol 83 (3) ◽  
pp. 518-533
Author(s):  
DIANE RIP ◽  
PIETER A. GOUWS
Keyword(s):  
South Africa ◽  
Listeria Monocytogenes ◽  
Restriction Fragment Length Polymorphism ◽  
Length Polymorphism ◽  
Food Processing ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Pcr Rflp ◽  
Restriction Fragment Length ◽  
Food Processing Environment

ABSTRACT Listeria monocytogenes is a ubiquitous, intracellular foodborne pathogen that is responsible for invasive listeriosis. The ability of L. monocytogenes to cause disease has some correlation with the serotypes of a specific lineage group, making the identification of lineage groups important for epidemiological analysis. The development of typing methods to link the strains of L. monocytogenes to an outbreak of listeriosis would help minimize the spread of the disease. The aim of this study was to design a PCR–restriction fragment length polymorphism (RFLP) method to differentiate between the lineage groups of L. monocytogenes. PCR-amplified fragments of the hly gene for 12 serotypes of L. monocytogenes were sequenced, aligned, and analyzed with the BioEdit program, and single nucleotide polymorphisms (SNPs) within regions of this gene were identified. Because of the difficulty in acquiring a serotype 4ab reference strain, this serotype was not included in this study. We tested the specificity and accuracy of the PCR-RFLP method on these L. monocytogenes reference strains and validated the method with 172 L. monocytogenes strains recovered from humans, food, and the food processing environment in 2000 to 2002 and 2008 to 2010 from regions within South Africa. PCR-RFLP analysis applied in this study placed L. monocytogenes serotypes into one of three lineage groups based on the sequence differences and SNPs within each lineage group. The SNPs were conserved in a region where RFLP analysis could be applied for a distinction between L. monocytogenes lineage groups. HIGHLIGHTS

scholarly journals Identification of Pneumococcal Serotypes by PCR–Restriction Fragment Length Polymorphism

Diagnostics ◽  
2019 ◽  
Vol 9 (4) ◽  
pp. 196 ◽  
Author(s):  
García-Suárez ◽  
González-Rodríguez ◽  
Cima-Cabal ◽  
Yuste ◽  
Vazquez ◽  
...  
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Dna Sequences ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Pcr Rflp ◽  
Restriction Fragment Length

Streptococcus pneumoniae shows more than 90 capsular serotypes that can be distinguished by their reactivity against antisera. The main objective of this work was the development of a molecular method for serotyping without the use of antisera. A computer program containing an algorithm was used to search in a database for potentially useful enzymes for Restriction Fragment Length Polymorphism-RFLP typing, in order to maximize the discrimination between different serotypes. DNA sequences of 90 serotypes for the region between dexB and aliA genes were compiled, and a computer screening of restriction enzymes was performed. The wzg–wzh–wzd–wze region and Sse9I restriction predicted unique PCR-RFLP patterns for 39 serotypes and eight serogroups. A second restriction enzyme resolved fragment specific patterns for 25 serotypes. The method was tested with 98 serotype-unknown clinical isolates. PCR-RFLP analysis deduced correct serotypes that were confirmed by Quellung reaction for 78.5% of the isolates.

scholarly journals Use of PCR-Restriction Fragment Length Polymorphism of inlA for Rapid Screening of Listeria monocytogenes Strains Deficient in the Ability To Invade Caco-2 Cells

2004 ◽  
Vol 70 (4) ◽  
pp. 2180-2185 ◽  
Author(s):  
S. Rousseaux ◽  
M. Olier ◽  
J. P. Lemaître ◽  
P. Piveteau ◽  
J. Guzzo
Keyword(s):  
Listeria Monocytogenes ◽  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Rapid Screening ◽  
Pcr Rflp ◽  
Reference Strains ◽  
Restriction Fragment Length

ABSTRACT A PCR-restriction fragment length polymorphism (RFLP) method was developed in order to screen a large number of strains for impaired adhesion to epithelial cells due to expression of truncated InlA. inlA polymorphism was analyzed by PCR-RFLP in order to correlate inlA PCR-RFLP profiles and production of truncated InlA. Thirty-seven Listeria monocytogenes strains isolated from various sources, including five noninvasive and two invasive reference strains, were screened. Two endonucleases (AluI and Tsp509I) were used, and they generated five composite profiles. Thirteen L. monocytogenes isolates were characterized by two specific PCR-RFLP profiles similar to PCR-RFLP profiles of noninvasive reference strains previously described as strains that produce truncated InlA. Ten of the 13 isolates showed low abilities to invade human epithelial Caco-2 cells. However, 4 of the 13 isolates were able to invade Caco-2 cells like reference strains containing complete InlA. Sequencing of inlA and Western blot analysis confirmed that truncated InlA was expressed in the 10 L. monocytogenes strains which were isolated from food. This PCR-RFLP method allowed us to identify 10 new strains expressing a truncated internalin. Based on the results obtained in this study, the PCR-RFLP method seems to be an interesting method for rapidly screening L. monocytogenes strains deficient in the ability to invade Caco-2 cells when a sizeable number of strains are studied.

scholarly journals Detection and Resolution of Cryptosporidium Species and Species Mixtures by Genus-Specific Nested PCR-Restriction Fragment Length Polymorphism Analysis, Direct Sequencing, and Cloning

2011 ◽  
Vol 77 (12) ◽  
pp. 3998-4007 ◽  
Author(s):  
Norma J. Ruecker ◽  
Rebecca M. Hoffman ◽  
Rachel M. Chalmers ◽  
Norman F. Neumann
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Nested Pcr ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Content Type ◽  
Pcr Rflp ◽  
Pcr Products ◽  
Restriction Fragment Length

ABSTRACTMolecular methods incorporating nested PCR-restriction fragment length polymorphism (RFLP) analysis of the 18S rRNA gene ofCryptosporidiumspecies were validated to assess performance based on limit of detection (LoD) and for detecting and resolving mixtures of species and genotypes within a single sample. The 95% LoD was determined for seven species (Cryptosporidium hominis,C. parvum,C. felis,C. meleagridis,C. ubiquitum,C. muris, andC. andersoni) and ranged from 7 to 11 plasmid template copies with overlapping 95% confidence limits. The LoD values for genomic DNA from oocysts on microscope slides were 7 and 10 template copies forC. andersoniandC. parvum, respectively. The repetitive nested PCR-RFLP slide protocol had an LoD of 4 oocysts per slide. When templates of two species were mixed in equal ratios in the nested PCR-RFLP reaction mixture, there was no amplification bias toward one species over another. At high ratios of template mixtures (>1:10), there was a reduction or loss of detection of the less abundant species by RFLP analysis, most likely due to heteroduplex formation in the later cycles of the PCR. Replicate nested PCR was successful at resolving many mixtures ofCryptosporidiumat template concentrations near or below the LoD. The cloning of nested PCR products resulted in 17% of the cloned sequences being recombinants of the two original templates. Limiting-dilution nested PCR followed by the sequencing of PCR products resulted in no sequence anomalies, suggesting that this method is an effective and accurate way to study the species diversity ofCryptosporidium, particularly for environmental water samples, in which mixtures of parasites are common.

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