Combined Loop-Mediated Isothermal Amplification Assays for Rapid Detection and One-Step Differentiation of Campylobacter jejuni and Campylobacter coli in Meat Products

A loop-mediated isothermal amplification (LAMP) assay system was established, allowing rplD gene-based simultaneous detection of Campylobacter jejuni and Campylobacter coli in enriched meat products. Additionally, one-step differentiation of target species on agar plates was enabled by cdtC gene- and gyrA gene-based duplex LAMP. Both the rplD and cdtC–gyrA LAMP assays amplified the target sequences in all 62 C. jejuni and 27 C. coli strains used for determining inclusivity and revealed 100% exclusivity toward 85 tested non-target species. Throughout the entire experiments, C. jejuni and C. coli strains were 100% distinguishable by melting curves of cdtC and gyrA LAMP products. After 24-h enrichment, the rplD LAMP assay reliably detected initial inoculation levels of 10–100 CFU/g in artificially contaminated minced meat. Investigation of naturally contaminated meat samples revealed a diagnostic accuracy of 95% toward real-time PCR and 94.1% toward the standard culture method applying the 24-h incubation period. Diagnostic sensitivity and specificity, and positive and negative predictive values were 89.8, 100, 100, and 91.2%, respectively, when measured against real-time PCR, and 89.6, 98.1, 97.7, and 91.2%, respectively, when measured against the standard culture method. After 48-h enrichment, the detection limit of the rplD LAMP assay improved to initial inoculation levels of 1–10 CFU/g in artificially contaminated minced meat. Applying the 48-h incubation period on naturally contaminated meat samples resulted in 100% concordant results between rplD LAMP, real-time PCR, and the standard culture method. The established LAMP assay system was proved to be suitable for rapid meat sample screening. Furthermore, it constitutes a promising tool for investigating other Campylobacter sources and could therefore make a valuable contribution to protect consumers from foodborne illness.

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Evaluation of StrepBSelectChromogenic Medium and the Fast-Track Diagnostics Group B Streptococcus (GBS) Real-Time PCR Assay Compared to Routine Culture for Detection of GBS during Antepartum Screening

Journal of Clinical Microbiology ◽

10.1128/jcm.00043-17 ◽

2017 ◽

Vol 55 (7) ◽

pp. 2137-2142 ◽

Cited By ~ 4

Author(s):

Deirdre L. Church ◽

Heather Baxter ◽

Tracie Lloyd ◽

Oscar Larios ◽

Daniel B. Gregson

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Fast Track ◽

Group B Streptococcus ◽

Culture Method ◽

Predictive Values ◽

Content Type ◽

Standard Culture ◽

Group B ◽

The Cost

ABSTRACTLife-threatening infection in neonates due to group BStreptococcus(GBS) is preventable by screening of near-term pregnant women and treatment at delivery. A total of 295 vaginal-rectal swabs were collected from women attending antepartum clinics in Calgary, Alberta, Canada. GBS colonization was detected by the standard culture method (Strep B Carrot Broth subcultured to blood agar with a neomycin disk) and compared to recovery with Strep Group B Broth (Dalynn Biologicals) subcultured to StrepBSelectchromogenic medium (CM; Bio-Rad Laboratories) and the Fast-Track Diagnostics GBS real-time PCR (quantitative PCR [qPCR]) assay (Phoenix Airmid Biomedical Corp.) performed with broth-enriched samples and the Abbottm2000sp/m2000rt system. A total of 62/295 (21%) women were colonized with GBS; 58 (19.7%) cases were detected by standard culture, while CM and qPCR each found 61 (20.7%) cases. The qPCR and CM were similar in performance, with sensitivities, specificities, and positive and negative predictive values of 98.4 and 98.4%, 99.6 and 99.6%, 98.4 and 98.4%, and 99.6 and 99.6%, respectively, compared to routine culture. Both qPCR and CM would allow more rapid reporting of routine GBS screening results than standard culture. Although the cost per test was similar for standard culture and CM, the routine use of qPCR would cost approximately four times as much as culture-based detection. Laboratories worldwide should consider implementing one of the newer methods for primary GBS testing, depending on the cost limitations of different health care jurisdictions.

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Development and evaluation of an ELIME assay to reveal the presence of Salmonella in irrigation water: Comparison with Real-Time PCR and the Standard Culture Method

Talanta ◽

10.1016/j.talanta.2015.11.015 ◽

2016 ◽

Vol 149 ◽

pp. 202-210 ◽

Cited By ~ 9

Author(s):

G. Volpe ◽

E. Delibato ◽

L. Fabiani ◽

E. Pucci ◽

S. Piermarini ◽

...

Keyword(s):

Real Time ◽

Irrigation Water ◽

Real Time Pcr ◽

Culture Method ◽

Standard Culture

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Comparison of PCR, Electrochemical Enzyme-Linked Immunosorbent Assays, and the Standard Culture Method for Detecting Salmonella in Meat Products

Applied and Environmental Microbiology ◽

10.1128/aem.70.3.1393-1396.2004 ◽

2004 ◽

Vol 70 (3) ◽

pp. 1393-1396 ◽

Cited By ~ 49

Author(s):

Luciana Croci ◽

Elisabetta Delibato ◽

Giulia Volpe ◽

Dario De Medici ◽

Giuseppe Palleschi

Keyword(s):

Immunosorbent Assay ◽

Flow Injection ◽

Flow Injection Analysis ◽

Meat Products ◽

Culture Method ◽

International Organization ◽

Enzyme Linked Immunosorbent Assay ◽

International Organization For Standardization ◽

Standard Culture ◽

Meat Samples

ABSTRACT An electrochemical enzyme-linked immunosorbent assay (ELISA) coupled with flow injection analysis (ELISA-FIA) and a PCR-based method using ST11 and ST15 primers for detecting salmonellae in meat were evaluated in comparison with the International Organization for Standardization (ISO) culture method. The methods were applied to experimentally contaminated and naturally contaminated meat samples. The results showed that both ELISA-FIA and PCR allowed detection of salmonella in a product contaminated with a low number of the microorganisms (1 to 10 salmonellae/25 g) after only 5 h of incubation of preenrichment broth, and they were just as effective as the ISO method.

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Comparative Studies of a Real-Time PCR Method and Three Enzyme-Linked Immunosorbent Assays for the Detection of Central Nervous System Tissues in Meat Products†

Journal of Food Protection ◽

10.4315/0362-028x-71.10.2059 ◽

2008 ◽

Vol 71 (10) ◽

pp. 2059-2066 ◽

Cited By ~ 3

Author(s):

HOLGER SCHÖNENBRÜCHER ◽

KATRIN ANNETTE GÖBEL ◽

AMIR ABDULMAWJOOD ◽

JÜRGEN A. RICHT ◽

MICHAEL BÜLTE

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Real Time ◽

Real Time Pcr ◽

Meat Products ◽

Spongiform Encephalopathy ◽

Pcr Assay ◽

Heat Treated ◽

Pcr Method ◽

Minced Meat

The removal of certain central nervous system (CNS) tissues (part of the bovine spongiform encephalopathy risk material) from the food chain is one of the highest priority tasks associated with avoiding contamination of the human food chain with the agent of bovine spongiform encephalopathy. A recently developed real-time PCR assay and three commercially available enzyme-linked immunosorbent assays (ELISAs) for the detection of CNS tissues in minced meat and three types of heat-treated sausages were evaluated. Bovine brain was used for spiking of internal reference material, and its detectability was examined during storage times of 12 months (for frozen minced meat and liver sausage) and 24 months (for sausages treated with medium and high heat). The real-time PCR method and both ELISA kits detected 0.1% CNS tissue in frozen minced meat and 0.1 or 1% CNS tissue in heat-treated meat products. The detectability of the amplified mRNA target region with the PCR assay was similar to the detectability of antigen by the ELISAs. Because the real-time PCR method also can be used to distinguish cattle, ovine, and caprine CNS tissues from porcine CNS tissues, it seems to be suitable as a routine diagnostic test for the sensitive and specific detection of CNS tissues in meat and meat products.

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Development of Conventional and Real-Time PCR Assays for the Rapid Detection of Group B Streptococci

Clinical Chemistry ◽

10.1093/clinchem/46.3.324 ◽

2000 ◽

Vol 46 (3) ◽

pp. 324-331 ◽

Cited By ~ 106

Author(s):

Danbing Ke ◽

Christian Ménard ◽

François J Picard ◽

Maurice Boissinot ◽

Marc Ouellette ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Rapid Detection ◽

Culture Method ◽

Conventional Pcr ◽

Pcr Assay ◽

Group B Streptococci ◽

Standard Culture ◽

Pcr Assays ◽

Group B

Abstract Background: Group B streptococci (GBS), or Streptococcus agalactiae, are the leading bacterial cause of meningitis and bacterial sepsis in newborns. Currently available rapid methods to detect GBS from clinical specimens are unsuitable for replacement of culture methods, mainly because of their lack of sensitivity. Methods: We have developed a PCR-based assay for the rapid detection of GBS. The cfb gene encoding the Christie-Atkins-Munch-Petersen (CAMP) factor was selected as the genetic target for the assay. The PCR primers were initially tested by a conventional PCR method followed by gel electrophoresis. The assay was then adapted for use with the LightCyclerTM. For this purpose, two fluorogenic adjacent hybridization probes complementary to the GBS-specific amplicon were designed and tested. In addition, a rapid sample-processing protocol was evaluated by colony-forming unit counting and PCR. A total of 15 vaginal samples were tested by both standard culture method and the two PCR assays. Results: The conventional PCR assay was specific because it amplified only GBS DNA among 125 bacterial and fungal species tested, and was able to detect all 162 GBS isolates from various geographical areas. This PCR assay allowed detection of as few as one genome copy of GBS. The real-time PCR assay was comparable to conventional PCR assay in terms of sensitivity and specificity, but it was more rapid, requiring only ∼30 min for amplification and computer-based data analysis. The presence of vaginal specimens had no detrimental effect on the sensitivity of the PCR with the sample preparation protocol used. All four GBS-positive samples identified by the standard culture method were detected by the two PCR assays. Conclusion: These assays provide promising tools for the rapid detection and identification of GBS.

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Real-time PCR High-resolution Melting Analysis for the Species Identification of Meat Products: Focusing on Food Safety and Detection of Meat Adulterations

Thrita ◽

10.5812/thrita.112550 ◽

2021 ◽

Vol In Press (In Press) ◽

Author(s):

Peyman Gholamnezhad ◽

Hamed Ahari ◽

Gholamreza Nikbakht Brujeni ◽

Seyed Amir Ali Anvar ◽

Abbas Ali Motalebi

Keyword(s):

High Resolution ◽

Real Time ◽

Species Identification ◽

Real Time Pcr ◽

High Resolution Melting ◽

Meat Products ◽

Hrm Analysis ◽

Melting Analysis ◽

Meat Species ◽

Meat Samples

Background: Real-time polymerase chain reaction (PCR) and high-resolution melting (HRM) analysis are currently considered as reliable techniques for the species identification of meat-based products and widely used to detect meat adulteration. Objectives: To examine the validity of real-time PCR and HRM analysis to identify meat species in meat-based products. Methods: Meat samples from five species (i.e., cattle, sheep, chicken, turkey, and wild pig) were purchased. Minced meat from the animal species of interest was prepared at the purities of 10%, and 20% and also were prepared as single and mixtures of two species. For molecular assessments, DNA samples were extracted from all the meat samples and subjected to real-time PCR by amplifying a mitochondrial cytochrome b specific for each species. Results: All the meat species studied in this research were successfully detected in the mixed meat samples when separately examined by real-time PCR. High-resolution melting analysis showed that all the meat species of interest were efficiently distinguished when examined simultaneously. Conclusions: The data presented here shows that the real-time PCR and HRM analysis are reliable methods for the identification of meat species used in meat products.

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Real-Time PCR Method Combined with a Matrix Lysis Procedure for the Quantification of Listeria monocytogenes in Meat Products

Foods ◽

10.3390/foods10040735 ◽

2021 ◽

Vol 10 (4) ◽

pp. 735

Author(s):

Mirian Labrador ◽

Carlota Giménez-Rota ◽

Carmen Rota

Keyword(s):

Listeria Monocytogenes ◽

Real Time ◽

Real Time Pcr ◽

Foodborne Pathogen ◽

Meat Products ◽

Reference Method ◽

Meat Industry ◽

Gene Copies ◽

Pcr Method ◽

Meat Samples

In this study a real-time PCR method has been developed for the specific quantification of the foodborne pathogen Listeria monocytogenes on meat products through the gene hlyA. The PCR was combined with a matrix lysis that allowed the obtaining of the microorganisms without sample dilution and the elimination the PCR inhibitors from dry-cured ham. The qPCR method calibration curve had an efficiency of 100.4%, limits of detection and quantification were 30.1 ± 6.2 CFU/g which is under the legal limit of L. monocytogenes in ready-to-eat products, and an analytical variability <0.25 log hlyA gene copies/reaction. The analysis was performed simultaneously with the reference method ISO 11290-2. The comparison of the qPCR-matrix lysis results with the reference method showed an excellent correspondence, with a relative accuracy between 95.83–105.20%. Finally, the method was applied to commercial derived meat samples and the pathogen was quantified in one of the commercial samples assayed in 69.1 ± 13.9 CFU/g while the reference method did not quantify it. The optimized qPCR showed higher precision and sensitivity than the reference method at low concentrations of the microorganism in a shorter time. Therefore, qPCR-matrix lysis shows a potential application in the meat industry for L. monocytogenes routine control.

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Evaluation of High Resolution Melting (HRM) Analysis for Meat Species Identification of Raw and Cooked Meat

Separations ◽

10.3390/separations8080116 ◽

2021 ◽

Vol 8 (8) ◽

pp. 116

Author(s):

Peyman Gholamnezhad ◽

Hamed Ahari ◽

Gholamreza Nikbakht Brujeni ◽

Seyed Amir Ali Anvar ◽

Abbasali Motallebi

Keyword(s):

High Resolution ◽

Real Time ◽

Species Identification ◽

Real Time Pcr ◽

High Resolution Melting ◽

Meat Products ◽

Pcr Assay ◽

Hrm Analysis ◽

Mitochondrial Cytochrome B ◽

Meat Samples

The current study aimed to examine a real-time PCR assay with high-resolution melting (HRM) analysis for the species identification of minced meat samples. Meat samples from several animal species were purchased and minced separately or as a mixture of two species. DNA was extracted from all meat samples and subjected to real-time PCR assay by amplifying species-specific mitochondrial cytochrome b regions. Regarding the meat mixtures, two separate melting curves with specific melt peak temperatures (Tm) were detected. Additionally, DNA from each species was quantified, based on the calibration curves. The results showed that a real-time PCR assay with HRM analysis is suitable for the species identification of meat products, and could be used for the detection of meat frauds.

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