scholarly journals Rapid identification of Salmonella serovars in feces by specific detection of virulence genes, invA and spvC, by an enrichment broth culture-multiplex PCR combination assay.

1996 ◽  
Vol 34 (10) ◽  
pp. 2619-2622 ◽  
Author(s):  
C H Chiu ◽  
J T Ou
Keyword(s):  
Multiplex Pcr ◽  
Virulence Genes ◽  
Rapid Identification ◽  
Broth Culture ◽  
Specific Detection ◽  
Enrichment Broth ◽  
Salmonella Serovars

scholarly journals Simultaneous Detection of Salmonella Strains and Escherichia coli O157:H7 with Fluorogenic PCR and Single-Enrichment-Broth Culture

2000 ◽  
Vol 66 (12) ◽  
pp. 5472-5476 ◽  
Author(s):  
Vijay K. Sharma ◽  
Steve A. Carlson
Keyword(s):  
Escherichia Coli ◽  
Simultaneous Detection ◽  
Broth Culture ◽  
Escherichia Coli O157 ◽  
Specific Detection ◽  
Pcr Assay ◽  
Enrichment Broth ◽  
E Coli ◽  
Serovar Typhimurium ◽  
Low Levels

ABSTRACT A multiplex fluorogenic PCR assay for simultaneous detection of pathogenic Salmonella strains and Escherichia coli O157:H7 was developed and evaluated for use in detecting very low levels of these pathogens in meat and feces. Two sets of primers were used to amplify a junctional segment of virulence genessipB and sipC of Salmonella and an intragenic segment of gene eae of E. coliO157:H7. Fluorogenic reporter probes were included in the PCR assay for automated and specific detection of amplified products. The assay could detect <10 CFU of Salmonella enterica serovar Typhimurium or E. coli O157:H7 per g of meat or feces artificially inoculated with these pathogens and cultured for 6 to 18 h in a single enrichment broth. Detection of amplification products could be completed in ≤4 h after enrichment.

Multiplex PCR Assay Simplifies Serotyping and Sequence Typing of Listeria monocytogenes Associated with Human Outbreaks

2005 ◽  
Vol 68 (9) ◽  
pp. 1907-1910 ◽  
Author(s):  
WEI ZHANG ◽  
STEPHEN J. KNABEL
Keyword(s):  
Listeria Monocytogenes ◽  
Multiplex Pcr ◽  
Gel Electrophoresis ◽  
Virulence Genes ◽  
Rapid Identification ◽  
Pulsed Field Gel Electrophoresis ◽  
Pcr Assay ◽  
Pulsed Field ◽  
Typing Scheme ◽  
Multiplex Pcr Assay

Listeria monocytogenes serotypes 1/2a and 4b are responsible for the majority of cases of human listeriosis worldwide. In this study, a multiplex PCR assay was developed to allow rapid identification and easily interpretable differentiation of serotypes 1/2a and 4b from other serotypes of L. monocytogenes by simultaneously targeting two virulence genes (inlB and inlC) and two serotype-specific genes (ORF2372 and lmo0171). A subsequent gel extraction and sequence typing analysis of the highly polymorphic intragenic regions in inlB and inlC simplified a previously developed multi–virulence-locus sequence typing scheme and provided discriminatory power for subtyping L. monocytogenes similar to pulsed-field gel electrophoresis analysis.

scholarly journals Use of an Enrichment Broth Cultivation-PCR Combination Assay for Rapid Diagnosis of Swine Erysipelas

1998 ◽  
Vol 36 (1) ◽  
pp. 86-89 ◽  
Author(s):  
Yoshihiro Shimoji ◽  
Yasuyuki Mori ◽  
Koji Hyakutake ◽  
Tsutomu Sekizaki ◽  
Yuichi Yokomizo
Keyword(s):  
Virulent Strain ◽  
Broth Culture ◽  
Erysipelothrix Rhusiopathiae ◽  
Specific Detection ◽  
First Line ◽  
Enrichment Broth ◽  
Dna Fragment ◽  
Conventional Methods ◽  
Swine Erysipelas ◽  
Highly Virulent

We have previously described the creation by Tn916mutagenesis of avirulent transposition mutants from a highly virulent strain of Erysipelothrix rhusiopathiae, the causative agent of swine erysipelas. In this study, we cloned a 2.2-kb DNA fragment which flanked the Tn916 insertion in an avirulent mutant (strain 33H6) and evaluated the possibility that this region could be used for the specific detection of E. rhusiopathiae. According to the sequences of this region, oligonucleotide primers were designed to amplify a 937-bp fragment of the E. rhusiopathiae chromosome by PCR. The specificity of the PCR was investigated by analyzing 64 strains of Erysipelothrixspecies and 27 strains of other genera different fromErysipelothrix. A 937-bp DNA fragment could be amplified from all E. rhusiopathiae strains tested, and no amplification was observed by using DNAs from the other species tested. To make a rapid and definite diagnosis of swine erysipelas in slaughterhouses, we developed an enrichment broth cultivation-PCR combination assay, which used a commercially available DNA extraction kit, to identify E. rhusiopathiae in the specimens from swine with arthritis. After samples were enriched in selective broth culture, detection of E. rhusiopathiae was tested by either conventional methods or the PCR. Of 102 samples tested, 15 samples were positive by conventional methods and 12 of the 15 samples were positive by the PCR. The detection limit of the PCR was 103 CFU per reaction mixture for the PCR-positive samples. These results indicate that this PCR technique could be used as a first-line screening technique for the specific detection of E. rhusiopathiae in specimens.

A Multiplex PCR Assay Mediated by Universal Primers for the Detection of Adulterated Meat in Mutton

2019 ◽  
Vol 82 (2) ◽  
pp. 325-330 ◽  
Author(s):  
WANWAN LIU ◽  
XIAONAN WANG ◽  
JING TAO ◽  
BANGSHENG XI ◽  
MAN XUE ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Detection System ◽  
Meat Products ◽  
Pcr Primers ◽  
Rapid Identification ◽  
Food Samples ◽  
Detection Sensitivity ◽  
Universal Primers ◽  
Multiplex Pcr Assay ◽  
Multiple Pcr

ABSTRACT This study aimed to establish a multiplex PCR detection system mediated by “universal primers,” which would be able to determine whether mutton meat contained nonmutton ingredients from rats, foxes, and ducks. Based on the sequence variation of specific mitochondrial genes, nine different multiplex PCR primers were designed, and four kinds of meat products were rapidly identified by electrophoresis using an optimized multiplex PCR system based on the molecular weight differences of the amplified products. Multiplex PCR applications optimized for meat food source from food samples for testing was used to verify the accuracy of the identification method. The results showed that the primers in multiple PCR system mediated by universal primers could be used for the rapid identification of rat, fox, duck, and sheep meat in mutton products, and the detection sensitivity could reach 0.05 ng/μL. The identification of food samples validated the practical value of this method. Therefore, a multiplex PCR system mediated by universal primers was established, which can be used to quickly identify the origin of animal ingredients from rats, foxes, and ducks in mutton products.

scholarly journals Rapid Identification, Differentiation, and Proposed New Taxonomic Classification of Bifidobacterium lactis

2002 ◽  
Vol 68 (12) ◽  
pp. 6429-6434 ◽  
Author(s):  
Marco Ventura ◽  
Ralf Zink
Keyword(s):  
Pcr Primers ◽  
Rapid Identification ◽  
Molecular Techniques ◽  
Rrna Gene ◽  
Specific Detection ◽  
Bifidobacterium Lactis ◽  
Bifidobacterium Animalis ◽  
Subspecies Level ◽  
Internally Transcribed Spacer

ABSTRACT Identification of Bifidobacterium lactis and Bifidobacterium animalis is problematic because of phenotypic and genetic homogeneities and has raised the question of whether they belong to one unique taxon. Analysis of the 16S-23S internally transcribed spacer region of B. lactis DSM10140T, B. animalis ATCC 25527T, and six potential B. lactis strains suggested two distinct clusters. Two specific 16S-23S spacer rRNA gene-targeted primers have been developed for specific detection of B. animalis. All of the molecular techniques used (B. lactis or B. animalis PCR primers, enterobacterial repetitive intergenic consensus PCR) demonstrated that B. lactis and B. animalis form two main groups and suggest a revision of the strains assigned to B. animalis. We propose that B. lactis should be separated from B. animalis at the subspecies level.

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 Effectiveness of Universal Pre-enrichment Broth for Recovery of Salmonella from Selected Dairy Foods

2003 ◽  
Vol 86 (4) ◽  
pp. 714-718 ◽  
Author(s):  
Thomas S Hammack ◽  
R Miguel Amaguaña ◽  
Mildred L Johnson ◽  
Wallace H Andrews
Keyword(s):  
Brilliant Green ◽  
The Other ◽  
Green Water ◽  
Enrichment Broth ◽  
Dairy Foods ◽  
Whole Milk ◽  
Dry Milk ◽  
Salmonella Serovars ◽  
Nonfat Dry Milk ◽  
Positive Results

Abstract The relative efficiencies of 2 Bacteriological Analytical Manual (BAM) pre-enrichments, lactose broth (LAC) and brilliant green water (BGW), were compared with Universal Pre-enrichment (UP) broth for the recovery of individual Salmonella serovars from instant nonfat dry milk (NFDM), dry whole milk (DWM), lactic casein (LC), and liquid whole milk (LWM). BGW was compared with UP broth for the analysis of NFDM and DWM but not with the other 2 matrixes. LAC was compared with UP broth for the analysis of LC and LWM. UP broth was made both from a commercial dehydrated preparation (UPC) and from individual ingredients (UPI). Bulk quantities of the selected dairy foods were inoculated with Salmonella serovars at levels intended to produce fractionally positive results, where at least half of the test portions analyzed, with one of the methods being evaluated, would be shown to be Salmonella-positive. For NFDM, in 6 of 9 experiments, with 2 different Salmonella serovars, BGW was significantly more productive than either UPI or UPC broth (p &lt; 0.05). Salmonella was recovered from 118 of 180 test portions with BGW, from 25 of 180 test portions with UPC, and from 14 of 180 test portions with UPI. For DWM, in 2 of 4 experiments, with 2 different Salmonella serovars, BGW was significantly more productive than either UPI or UPC broth (p &lt; 0.05). Salmonella was recovered from 67 of 80 test portions with BGW, from 36 of 80 test portions with UPC, and from 37 of 80 test portions with UPI. For LWM, in 9 of 9 experiments, with 3 different Salmonella serovars, there were no significant differences among the broths. Salmonella was recovered from 120 of 180 test portions with LAC, from 135 of 180 test portions with UPC, and from 129 of 180 test portions with UPI. For LC, in 5 of 7 experiments, with 2 different Salmonella serovars, both UPI and UPC broth were significantly more productive than LAC (p &lt; 0.05). Salmonella was recovered from 42 of 140 test portions with LAC, from 114 of 140 test portions with UPC, and from 114 of 140 test portions with UPI. In addition, overall results showed that UPC and UPI broths were equivalent for the recovery of Salmonella from the foods tested, without regard to their performance in comparison with either LAC or BGW.

scholarly journals Strain-specific detection of two Aureobasidium pullulans strains, fungal biocontrol agents of fire blight by new, developed multiplex-PCR

2008 ◽  
Vol 104 (5) ◽  
pp. 1433-1441 ◽  
Author(s):  
I. Loncaric ◽  
C. Donat ◽  
B. Antlinger ◽  
J.T. Oberlerchner ◽  
B. Heissenberger ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Aureobasidium Pullulans ◽  
Fire Blight ◽  
Biocontrol Agents ◽  
Specific Detection
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