An Improved PCR Primer Pair Based on 16S rDNA for the Specific Detection of Salmonella Serovars in Food Samples

2004 ◽  
Vol 67 (7) ◽  
pp. 1335-1343 ◽  
Author(s):  
CHIEN-KU LIN ◽  
CHO-LIEN HUNG ◽  
SHU-CHEN HSU ◽  
CHENG-CHIH TSAI ◽  
HAU-YANG TSEN
Keyword(s):  
Sequence Data ◽  
Complete Sequence ◽  
Food Samples ◽  
Chicken Meat ◽  
Rrna Gene ◽  
Whole Milk ◽  
Sporadic Cases ◽  
Salmonella Serovars ◽  
Primer Annealing ◽  
Pcr Primer

Salmonella serovars are some of the major bacterial pathogens that can cause sporadic cases and outbreaks of foodborne illness. Based on the sequence data in the V3 region of the 16S rRNA gene, two PCR primer pairs have been designed for the detection of all serovars of Salmonella. However, none of these primers were specific for Salmonella because complete sequence homology with certain non-Salmonella strains has been found within each of them. Thus, the specificities of these two primer pairs could not rely on only one of the two primers. In this study, we modified our previous 16SFI primer by extending one base at the 5′ end and three bases at the 3′ end. The modified primer, 16S-Sal, was designed with one or more mismatched bases near the 3′ end of the primer annealing to the corresponding sequences of non-Salmonella strains. Such modification eliminates interference from Citrobacter freundii and Enterobacter cloacae as occurs with the 16SFI primer. When 16S-Sal and a degenerate primer, 16S-CCR, were used as a primer pair, detection specificity of Salmonella serovars was achieved. Because this primer pair was used for PCR detection of the salmonellae in food samples, such as whole milk and chicken meat, as low as 1 to 9 CFU/g (ml) of the food sample could be detected when a 8-h preculture step was performed prior to the PCR. For chicken meat, the endogenous microflora did not interfere with the PCR results.

scholarly journals Detection ofYersinia enterocoliticain Retail Chicken Meat, Mashhad, Iran

2018 ◽  
Vol 2018 ◽  
pp. 1-4 ◽  
Author(s):  
Khadigeh Sirghani ◽  
Tayebeh Zeinali ◽  
Abdollah Jamshidi
Keyword(s):  
Yersinia Enterocolitica ◽  
Health Hazard ◽  
Culture Method ◽  
Good Alternative ◽  
Food Samples ◽  
Selective Medium ◽  
Chicken Meat ◽  
Poultry Meat ◽  
Rrna Gene ◽  
Pcr Assay

Poultry meat is one of the most important sources of infection ofYersiniaspp. for humans. The aim of the present study was to evaluate the incidence ofYersinia enterocoliticain chicken meat by using culture method on selective medium and confirmation by PCR assay. Also, biochemical methods were used for biotyping. A total of 100 chicken thigh meat samples were collected randomly from retail outlets in Mashhad, Iran. Samples were enriched in Peptone-Sorbitol-Bile (PSB) broth and then cultured on Cefsulodin-Irgasan-Novobiocin (CIN) agar containing antibiotics supplement. The DNA was extracted from suspected colonies ofYersiniaspp. and then PCR test using specific primers for 16S rRNA gene ofYersinia enterocoliticawas performed. In this study, 30% of chicken meat was contaminated withYersiniaspp. by culture method and 25% of chicken meat was contaminated withYersinia enterocolitica. Biotyping of isolated colonies showed that all of the isolates belonged to biotype 1A. Culture and detection ofYersiniaspp. from food samples traditionally take 4 days. Due to high accuracy and speed of PCR assay, it is a good alternative method for microbiological techniques. In conclusion, poultry meat can act as a source ofY. enterocoliticaand could be considered as a public health hazard.

PCR Detection of Alternaria spp. in Processed Foods, Based on the Internal Transcribed Spacer Genetic Marker

2011 ◽  
Vol 74 (2) ◽  
pp. 240-247 ◽  
Author(s):  
MIGUELÁNGEL PAVÓN ◽  
ISABEL GONZÁLEZ ◽  
MARÍA ROJAS ◽  
NICOLETTE PEGELS ◽  
ROSARIO MARTÍN ◽  
...  
Keyword(s):  
Food Processing ◽  
Internal Transcribed Spacer ◽  
Rapid Identification ◽  
Food Samples ◽  
Pcr Analysis ◽  
Infant Food ◽  
Rrna Gene ◽  
Tomato Pulp ◽  
Pcr Method ◽  
Fungal Contaminants

The genus Alternaria is considered one of the most important fungal contaminants of vegetables, fruits, and cereals, producing several mycotoxins that can withstand food processing methods. Conventional methods for Alternaria identification and enumeration are laborious and time-consuming, and they might not detect toxigenic molds inactivated by food processing. In this study, a PCR method has been developed for the rapid identification of Alternaria spp. DNA in foodstuffs, based on oligonucleotide primers targeting the internal transcribed spacer (ITS) 1 and ITS2 regions of the rRNA gene. The specificity of the Alternaria-specific primer pair designed (Dir1ITSAlt–Inv1ITSAlt) was verified by PCR analysis of DNA from various Alternaria spp., and also from several fungal, bacterial, yeast, animal, and plant species. The detection limit of the method was 102 CFU/ml in viable culture, heated culture, or experimentally inoculated tomato pulp. The applicability of the method for detection of Alternaria spp. DNA in foodstuffs was assessed by testing several commercial samples. Alternaria DNA was detected in 100% of spoiled tomato samples, 8% of tomato products, and 36.4% of cereal-based infant food samples analyzed.

Ultrasensitive determination of chloramphenicol in pork and chicken meat samples using a portable electrochemical sensor: effects of 2D nanomaterials on the sensing performance and stability

2021 ◽  
Author(s):  
Ngo Xuan Dinh ◽  
Tuyet Nhung Pham ◽  
Tran Quang Huy ◽  
Do Quang Trung ◽  
Pham Anh Tuan ◽  
...  
Keyword(s):  
Electrochemical Sensor ◽  
Food Samples ◽  
Chicken Meat ◽  
Electrochemical Sensing ◽  
Portable Sensors ◽  
2D Nanomaterials ◽  
Meat Samples ◽  
Sensing Performance

This work contributes to a deeper understanding of the effects of functional 2D nanomaterials on the electrochemical sensing performance of SPE-based portable sensors for the rapid, accurate, and on-site determination of CAP in food samples.

scholarly journals Introducing mothur: Open-Source, Platform-Independent, Community-Supported Software for Describing and Comparing Microbial Communities

2009 ◽  
Vol 75 (23) ◽  
pp. 7537-7541 ◽  
Author(s):  
Patrick D. Schloss ◽  
Sarah L. Westcott ◽  
Thomas Ryabin ◽  
Justine R. Hall ◽  
Martin Hartmann ◽  
...  
Keyword(s):  
16S Rrna ◽  
Software Package ◽  
Sequence Data ◽  
Rrna Gene ◽  
Sequencing Data ◽  
Laptop Computer ◽  
Operational Taxonomic Units ◽  
Β Diversity ◽  
Single Piece

ABSTRACT mothur aims to be a comprehensive software package that allows users to use a single piece of software to analyze community sequence data. It builds upon previous tools to provide a flexible and powerful software package for analyzing sequencing data. As a case study, we used mothur to trim, screen, and align sequences; calculate distances; assign sequences to operational taxonomic units; and describe the α and β diversity of eight marine samples previously characterized by pyrosequencing of 16S rRNA gene fragments. This analysis of more than 222,000 sequences was completed in less than 2 h with a laptop computer.

Integrating genomics into the taxonomy and systematics of the Bacteria and Archaea

2014 ◽  
Vol 64 (Pt_2) ◽  
pp. 316-324 ◽  
Author(s):  
Jongsik Chun ◽  
Fred A. Rainey
Keyword(s):  
Genomic Sequence ◽  
Sequence Data ◽  
Original Research ◽  
Rrna Gene ◽  
New Taxon ◽  
Genome Sequences ◽  
Microbial World ◽  
Content Type ◽  
Link Type ◽  
Type Strains

The polyphasic approach used today in the taxonomy and systematics of the Bacteria and Archaea includes the use of phenotypic, chemotaxonomic and genotypic data. The use of 16S rRNA gene sequence data has revolutionized our understanding of the microbial world and led to a rapid increase in the number of descriptions of novel taxa, especially at the species level. It has allowed in many cases for the demarcation of taxa into distinct species, but its limitations in a number of groups have resulted in the continued use of DNA–DNA hybridization. As technology has improved, next-generation sequencing (NGS) has provided a rapid and cost-effective approach to obtaining whole-genome sequences of microbial strains. Although some 12 000 bacterial or archaeal genome sequences are available for comparison, only 1725 of these are of actual type strains, limiting the use of genomic data in comparative taxonomic studies when there are nearly 11 000 type strains. Efforts to obtain complete genome sequences of all type strains are critical to the future of microbial systematics. The incorporation of genomics into the taxonomy and systematics of the Bacteria and Archaea coupled with computational advances will boost the credibility of taxonomy in the genomic era. This special issue of International Journal of Systematic and Evolutionary Microbiology contains both original research and review articles covering the use of genomic sequence data in microbial taxonomy and systematics. It includes contributions on specific taxa as well as outlines of approaches for incorporating genomics into new strain isolation to new taxon description workflows.

scholarly journals Taxonomic characterization of nine strains isolated from clinical and environmental specimens, and proposal of Corynebacterium tuberculostearicum sp. nov.

2004 ◽  
Vol 54 (4) ◽  
pp. 1055-1061 ◽  
Author(s):  
Carole Feurer ◽  
Dominique Clermont ◽  
François Bimet ◽  
Adina Candréa ◽  
Mary Jackson ◽  
...  
Keyword(s):  
Gene Sequence ◽  
Fatty Acid Content ◽  
Food Samples ◽  
Cell Protein ◽  
Rrna Gene ◽  
Biochemical Tests ◽  
Taxonomic Analysis ◽  
Gene Sequence Analysis ◽  
Taxonomic Characterization

Nine unidentified Gram-positive, lipophilic corynebacteria were isolated from clinical and food samples and subjected to a polyphasic taxonomic analysis. The bacteria were distinguished from Corynebacterium species with validly published names by biochemical tests, fatty acid content and whole-cell protein analysis. Comparative 16S rRNA gene sequence analysis demonstrated unambiguously that the nine strains were related phylogenetically to the species ‘Corynebacterium tuberculostearicum’ and represented a distinct subline within the genus Corynebacterium. On the basis of both phenotypic and phylogenetic evidence, the formal description of Corynebacterium tuberculostearicum sp. nov. is proposed. The type strain of C. tuberculostearicum is Medalle XT (=LDC-20T=CIP 107291T=CCUG 45418T=ATCC 35529T).

Detection of Bacillus Spores Using PCR and FTA Filters

2004 ◽  
Vol 67 (5) ◽  
pp. 1036-1038 ◽  
Author(s):  
KEITH A. LAMPEL ◽  
DEANNE DYER ◽  
LEROY KORNEGAY ◽  
PALMER A. ORLANDI
Keyword(s):  
Nested Pcr ◽  
Pcr Primers ◽  
Food Samples ◽  
Microbial Pathogens ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Conserved Sequence ◽  
Bacillus Spp ◽  
Bacillus Spores ◽  
Template Dna

Emphasis has been placed on developing and implementing rapid detection systems for microbial pathogens. We have explored the utility of expanding FTA filter technology for the preparation of template DNA for PCR from bacterial spores. Isolated spores from several Bacillus spp., B. subtilis, B. cereus, and B. megaterium, were applied to FTA filters, and specific DNA products were amplified by PCR. Spore preparations were examined microscopically to ensure that the presence of vegetative cells, if any, did not yield misleading results. PCR primers SRM86 and SRM87 targeted a conserved region of bacterial rRNA genes, whereas primers Bsub5F and Bsub3R amplified a product from a conserved sequence of the B. subtilis rRNA gene. With the use of the latter set of primers for nested PCR, the sensitivity of the PCR-based assay was increased. Overall, 53 spores could be detected after the first round of PCR, and the sensitivity was increased to five spores by nested PCR. FTA filters are an excellent platform to remove PCR inhibitors and have universal applications for environmental, clinical, and food samples.

A new species of trypanosome, Trypanosoma desterrensis sp. n., isolated from South American bats

Parasitology ◽  
2003 ◽  
Vol 127 (3) ◽  
pp. 265-271 ◽  
Author(s):  
E. C. GRISARD ◽  
N. R. STURM ◽  
D. A. CAMPBELL
Keyword(s):  
New Species ◽  
Dna Sequence ◽  
Sequence Data ◽  
Rrna Gene ◽  
South American ◽  
Sequence Variability ◽  
Dna Sequence Data ◽  
Hybridization Probes ◽  
5S Rrna Gene ◽  
A New Species

Trypanosomes isolated from South American bats include the human pathogen Trypanosoma cruzi. Other Trypanosoma spp. that have been found exclusively in bats are not well characterized at the DNA sequence level and we have therefore used the SL RNA gene to differentiate and characterize kinetoplastids isolated from bats in South America. A Trypanosoma sp. isolated from bats in southern Brazil was compared with the geographically diverse isolates T. cruzi marinkellei, T. vespertilionis, and T. dionisii. Analysis of the SL RNA gene repeats revealed size and sequence variability among these bat trypanosomes. We have developed hybridization probes to separate these bat isolates and have analysed the DNA sequence data to estimate their relatedness. A new species, Trypanosoma desterrensis sp. n., is proposed, for which a 5S rRNA gene was also found within the SL RNA repeat.

scholarly journals Anomalous placement of introns in a member of the intermediate filament multigene family: an evolutionary conundrum.

1986 ◽  
Vol 6 (5) ◽  
pp. 1529-1534 ◽  
Author(s):  
S A Lewis ◽  
N J Cowan
Keyword(s):  
Glial Fibrillary Acidic Protein ◽  
Intermediate Filament ◽  
Multigene Family ◽  
Protein Gene ◽  
Sequence Data ◽  
Complete Sequence ◽  
Acidic Protein ◽  
Type I ◽  
Neurofilament Protein ◽  
Gene Encoding

The origin of introns and their role (if any) in gene expression, in the evolution of the genome, and in the generation of new expressed sequences are issues that are understood poorly, if at all. Multigene families provide a favorable opportunity for examining the evolutionary history of introns because it is possible to identify changes in intron placement and content since the divergence of family members from a common ancestral sequence. Here we report the complete sequence of the gene encoding the 68-kilodalton (kDa) neurofilament protein; the gene is a member of the intermediate filament multigene family that diverged over 600 million years ago. Five other members of this family (desmin, vimentin, glial fibrillary acidic protein, and type I and type II keratins) are encoded by genes with six or more introns at homologous positions. To our surprise, the number and placement of introns in the 68-kDa neurofilament protein gene were completely anomalous, with only three introns, none of which corresponded in position to introns in any characterized intermediate filament gene. This finding was all the more unexpected because comparative amino acid sequence data suggest a closer relationship of the 68-kDa neurofilament protein to desmin, vimentin, and glial fibrillary acidic protein than between any of these three proteins and the keratins. It appears likely that an mRNA-mediated transposition event was involved in the evolution of the 68-kDa neurofilament protein gene and that subsequent events led to the acquisition of at least two of the three introns present in the contemporary sequence.

scholarly journals Epipelic cyanobacterial diversity in Pinang River basin, Malaysia, revealed by 16S-based metagenomic approach

Algologia ◽  
2021 ◽  
Vol 31 (1) ◽  
pp. 93-113
Author(s):  
A.R. Nur Fadzliana ◽  
◽  
W.O. Wan Maznah ◽  
S.A.M. Nor ◽  
Choon Pin Foong ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
River Basin ◽  
Sequence Data ◽  
Diversity Index ◽  
Saline Water ◽  
Illumina Miseq ◽  
Rrna Gene ◽  
Cyanobacterial Community ◽  
Upstream Station

Cyanobacteria are the most widespread group of photosynthetic prokaryotes. They are primary producers in a wide variety of habitats and are able to thrive in harsh environments, including polluted waters; therefore, this study was conducted to explore the cyanobacterial populations inhabiting river tributaries with different levels of pollution. Sediment samples (epipelon) were collected from selected tributaries of the Pinang River basin. Air Terjun (T1) and Air Itam rivers (T2) represent the upper streams of Pinang River basin, while Dondang (T3) and Jelutong rivers (T4) are located at in the middle of the river basin. The Pinang River (T5) is located near the estuary and is subjected to saline water intrusion during high tides. Cyanobacterial community was determined by identifying the taxa via 16S rRNA gene amplicon sequence data. 16S rRNA gene amplicons generated from collected samples were sequenced using illumina Miseq, with the targeted V3 and V4 regions yielding approximately 1 mln reads per sample. Synechococcus, Phormidium, Arthronema and Leptolyngbya were found in all samples. Shannon-Weiner diversity index was highest (H’ = 1.867) at the clean upstream station (T1), while the moderately polluted stream (T3) recorded the lowest diversity (H’ = 0.399), and relatively polluted stations (T4 and T5) recorded fairly high values of H’. This study provides insights into the cyanobacterial community structure in Pinang River basin via cultivation-independent techniques using 16S rRNA gene amplicon sequence. Occurrence of some morphospecies at specific locations showed that the cyanobacterial communities are quite distinct and have specific ecological demands. Some species which were ubiquitous might be able to tolerate varied environmental conditions.

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