Real-Time PCR Detection of the Thermostable Direct Hemolysin and Thermolabile Hemolysin Genes in a Vibrio parahaemolyticus Cultured from Mussels and Mussel Homogenate Associated with a Foodborne Outbreak

Molecular methods have become vital epidemiological tools in the detection and characterization of bacteria associated with a foodborne outbreak. We used both culture and real-time PCR to detect a Vibrio parahaemolyticus isolate associated with a foodborne outbreak. The outbreak occurred in July 2002 in Polk County, Florida, and originated at a Chinese buffet, with one person being hospitalized. The hospital isolated V. parahaemolyticus from the patient but destroyed the sample after diagnosis. From an onsite visit of the restaurant, food samples that possibly contributed to the outbreak were collected and sent to the Florida Department of Health, Tampa Branch Laboratory. Crab legs, crabsticks, and mussel samples were homogenized and incubated according to the Food and Drug Administration Bacteriological Analytical Manual culture protocol. Three sets of primers and a TaqMan probe were designed to target the tdh, trh, and tlh genes and used for real-time PCR. This study was successful in isolating V. parahaemolyticus from a mussel sample and detecting two of its genes (tdh and tlh) in food and pure culture by real-time PCR.

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Rapid Detection of Salmonella in Foods Using Real-Time PCR

Journal of Food Protection ◽

10.4315/0362-028x-71.12.2436 ◽

2008 ◽

Vol 71 (12) ◽

pp. 2436-2441 ◽

Cited By ~ 77

Author(s):

CHORNG-MING CHENG ◽

WEN LIN ◽

KHANH THIEN VAN ◽

LIEUCHI PHAN ◽

NELLY N. TRAN ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Rapid Detection ◽

Food Samples ◽

Taqman Probe ◽

Cell Culturing ◽

Chili Powder ◽

Pcr Method ◽

Food Matrices ◽

Salmonella Serovars

Conventional methods for detection of Salmonella serovars in foods are generally time-consuming and labor intensive. A real-time PCR method has been developed with custom designed primers and a TaqMan probe to detect the presence of a 262-bp fragment of the Salmonella-specific invA gene. The method has been tested with a total of 384 field-isolated Salmonella serovars and non-Salmonella stock strains, as well as 420 U.S. Food and Drug Administration food samples, comprising a variety of food matrices. The method was highly specific in detecting Salmonella in spiked chili powder and shrimp samples, with a sensitivity of 0.04 CFU/g. In addition, the method is faster, more accurate, and less costly than the traditional U.S. Food and Drug Administration's Bacteriological Analytical Manual cell-culturing and the AOAC International–approved VIDAS methods to detect Salmonella in foods.

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Molecular Characterization of Giardia duodenalis in Children and Adults Sampled in Algeria

Microorganisms ◽

10.3390/microorganisms9010054 ◽

2020 ◽

Vol 9 (1) ◽

pp. 54

Author(s):

Salem Belkessa ◽

Daniel Thomas-Lopez ◽

Karim Houali ◽

Farida Ghalmi ◽

Christen Rune Stensvold

Keyword(s):

Real Time ◽

Molecular Characterization ◽

Real Time Pcr ◽

Intestinal Parasites ◽

Giardia Duodenalis ◽

Specific Pcr ◽

Stool Samples ◽

Pcr Targeting ◽

Assemblage B

The molecular epidemiology of giardiasis in Africa remains unclear. A study was carried out across four hospitals in Algeria. A total of 119 fecal samples from 55 children, 37 adults, and 27 individuals of undetermined age, all scored positive for intestinal parasites by microscopy, and were screened by real-time PCR for Giardia. Molecular characterization of Giardia was performed by assemblage-specific PCR and PCR targeting the triose phosphate isomerase gene (tpi). Of the 119 samples, 80 (67%) were Giardia-positive by real-time PCR. For 48 moderately-highly real-time PCR-positive samples, tpi genotyping assigned 22 samples to Assemblage A and 26 to Assemblage B. Contrary to Assemblage A, Assemblage B exhibited substantial genetic diversity and allelic heterozygosity. Assemblage-specific PCR proved to be specific for discriminating Assemblage A or B but not as sensitive as tpi genotyping. We confirmed that real-time PCR is more sensitive than microscopy for detecting Giardia in stool samples and that robust amplification and sequencing of the tpi gene is feasible when moderate-to-strongly real-time PCR-positive samples are used. This study is one of the few performed in Africa providing genotyping data on Giardia infections in humans. Both assemblages A and B were commonly seen and not associated with specific sociodemographic data.

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Development of Rapid Real-Time PCR and Most-Probable-Number Real-Time PCR Assays To Quantify Enterotoxigenic Strains of the Species in the Bacillus cereus Group

Journal of Food Protection ◽

10.4315/0362-028x-70.12.2774 ◽

2007 ◽

Vol 70 (12) ◽

pp. 2774-2781 ◽

Cited By ~ 17

Author(s):

I-CHEN YANG ◽

DANIEL YANG-CHIH SHIH ◽

JAN-YI WANG ◽

TZU-MING PAN

Keyword(s):

Bacillus Cereus ◽

Real Time ◽

Real Time Pcr ◽

Food Samples ◽

Most Probable Number ◽

Pcr Assay ◽

Bacillus Cereus Group ◽

Probable Number ◽

Cooked Rice ◽

Group Detection

Members of the Bacillus cereus group may produce diarrheal enterotoxins and could be potential hazards if they enter the food chain. Therefore, a method capable of detecting all the species in the B. cereus group rather than B. cereus alone is important. We selected nhe as the target and developed a real-time PCR assay to quantify enterotoxigenic strains of the B. cereus group. The real-time PCR assay was evaluated with 60 B. cereus group strains and 28 others. The assay was also used to construct calibration curves for different food matrices and feces. The assay has an excellent quantification capacity, as proved by its linearity (R2 > 0.993), wide dynamic quantification range (102 to 107 CFU/g for cooked rice and chicken, 103 to 107 CFU/ml for milk, and 104 to 107 CFU/g for feces), and adequate relative accuracy (85.5 to 101.1%). For the low-level contaminations, a most-probable-number real-time PCR assay was developed that could detect as low as 100 CFU/ml. Both assays were tested with real food samples and shown to be considerably appropriate for B. cereus group detection and quantification.

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Multiplex Real-Time PCR Method for Simultaneous Identification and Toxigenic Type Characterization of Clostridium difficile From Stool Samples

Annals of Laboratory Medicine ◽

10.3343/alm.2015.35.3.306 ◽

2015 ◽

Vol 35 (3) ◽

pp. 306-313 ◽

Cited By ~ 10

Author(s):

Abdullah Kilic ◽

Mohammad J. Alam ◽

Naradah L. Tisdel ◽

Dhara N. Shah ◽

Mehmet Yapar ◽

...

Keyword(s):

Clostridium Difficile ◽

Real Time ◽

Real Time Pcr ◽

Stool Samples ◽

Pcr Method ◽

Simultaneous Identification

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Real-time PCR von virulenten Vibrio parahaemolyticus in Fisch- und Krebstierprodukten

Journal of Consumer Protection and Food Safety ◽

10.1007/s00003-007-0182-y ◽

2007 ◽

Vol 2 (2) ◽

pp. 213-217 ◽

Cited By ~ 1

Author(s):

A. Lehmacher ◽

B. Hansen

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Vibrio Parahaemolyticus

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Characterization of Plasmodium ovale curtisi and P. ovale wallikeri in Western Kenya Utilizing a Novel Species-specific Real-time PCR Assay

PLoS Neglected Tropical Diseases ◽

10.1371/journal.pntd.0003469 ◽

2015 ◽

Vol 9 (1) ◽

pp. e0003469 ◽

Cited By ~ 16

Author(s):

Robin H. Miller ◽

Clifford O. Obuya ◽

Elizabeth W. Wanja ◽

Bernhards Ogutu ◽

John Waitumbi ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Novel Species ◽

Plasmodium Ovale ◽

Pcr Assay ◽

Western Kenya ◽

Plasmodium Ovale Curtisi ◽

Species Specific

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Molecular characterization and bioinformatics analysis of Ncoa7B, a novel ovulation-associated and reproduction system-specific Ncoa7 isoform

Reproduction ◽

10.1530/rep-07-0402 ◽

2008 ◽

Vol 135 (3) ◽

pp. 321-333 ◽

Cited By ~ 14

Author(s):

Ketty Shkolnik ◽

Shifra Ben-Dor ◽

Dalia Galiani ◽

Ariel Hourvitz ◽

Nava Dekel

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Oxidative Dna Damage ◽

Bioinformatics Analysis ◽

Cdna Clones ◽

Nuclear Receptor Coactivator ◽

Reproduction System ◽

Preovulatory Follicles ◽

Multiple Signaling

In the present work, we employed bioinformatics search tools to select ovulation-associated cDNA clones with a preference for those representing putative novel genes. Detailed characterization of one of these transcripts, 6C3, by real-time PCR and RACE analyses led to identification of a novel ovulation-associated gene, designatedNcoa7B. This gene was found to exhibit a significant homology to theNcoa7gene that encodes a conserved tissue-specific nuclear receptor coactivator. UnlikeNcoa7,Ncoa7Bpossesses a unique and highly conserved exon at the 5′ end and encodes a protein with a unique N-terminal sequence. Extensive bioinformatics analysis has revealed thatNcoa7Bhas one identifiable domain, TLDc, which has recently been suggested to be involved in protection from oxidative DNA damage. An alignment of TLDc domain containing proteins was performed, and the closest relative identified wasOXR1, which also has a corresponding, highly related short isoform, with just a TLDc domain. Moreover,Ncoa7Bexpression, as seen to date, seems to be restricted to mammals, while other TLDc family members have no such restriction. Multiple tissue analysis revealed that unlikeNcoa7, which was abundant in a variety of tissues with the highest expression in the brain,Ncoa7BmRNA expression is restricted to the reproductive system organs, particularly the uterus and the ovary. The ovarian expression ofNcoa7Bwas stimulated by human chorionic gonadotropin. Additionally, using real-time PCR, we demonstrated the involvement of multiple signaling pathways forNcoa7Bexpression on preovulatory follicles.

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Isolation and molecular characterization of Mycobacterium tuberculosis complex isolated from raw milk in some dairy farms in Egypt

International Journal of Basic and Applied Sciences ◽

10.14419/ijbas.v5i2.5299 ◽

2016 ◽

Vol 5 (2) ◽

pp. 105

Author(s):

Heba Hussien ◽

Eman Mahrous

Keyword(s):

Mycobacterium Tuberculosis ◽

Real Time ◽

Real Time Pcr ◽

Culture Method ◽

Raw Milk ◽

Accurate Method ◽

Tuberculosis Complex ◽

Milk Samples ◽

Source Of Infection

This study was conducted to detect Mycobacterium tuberculosis complex in milk in three Egyptian Governorates; El-Sharkia, El-Menoufia and El-Behera Governorates. 300 milk samples were collected from tuberculin positive cases, 18 (6.0%) were shedding Mycobacterium tuberculosis complex in their milk which detected by real time PCR. On another hand, 170 milk samples were collected from tuberculin negative cases, 5 (2.9%) were shedding Mycobacterium tuberculosis complex in their milk which detected by real time PCR. All milk samples were examined by three techniques including Microscopic examination, culture and real time PCR. Real time PCR is more rapid and accurate method than microscopic and culture method. The isolated colonies from culture were examined by Multiplex PCR to demonstrate the source of infection either human or animal source.

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