Specific PCR Detection of Arcobacter butzleri, Arcobacter cryaerophilus, Arcobacter skirrowii, and Arcobacter cibarius in Chicken Meat

2009 ◽  
Vol 72 (7) ◽  
pp. 1491-1495 ◽  
Author(s):  
DANIELA PENTIMALLI ◽  
NICOLETTE PEGELS ◽  
TERESA GARCÍA ◽  
ROSARIO MARTÍN ◽  
ISABEL GONZÁLEZ
Keyword(s):  
Pcr Amplification ◽  
Chicken Meat ◽  
Pcr Analysis ◽  
Rrna Gene ◽  
Pcr Assay ◽  
Gyra Gene ◽  
Specific Primers ◽  
Specific Pcr ◽  
Arcobacter Butzleri ◽  
Species Specific

An enrichment PCR assay using species-specific primers was developed for the detection of Arcobacter butzleri, Arcobacter cryaerophilus, Arcobacter skirrowii, and Arcobacter cibarius in chicken meat. Primers for A. cryaerophilus, A. skirrowii, and A. cibarius were designed based on the gyrA gene to amplify nucleic acid fragments of 212, 257, and 145 bp, respectively. The A. butzleri–specific primers were designed flanking a 203-bp DNA fragment in the 16S rRNA gene. The specificity of the four primer pairs was assessed by PCR analysis of DNA from a panel of Arcobacter species, related Campylobacter, Helicobacter species, and other food bacteria. The applicability of the method was then validated by testing 42 fresh retail-purchased chicken samples in the PCR assay. An 18-h selective preenrichment step followed by PCR amplification with the four Arcobacter primer sets revealed the presence of Arcobacter spp. in 85.7% of the retail chicken samples analyzed. A. butzleri was the only species present in 50% of the samples, and 35.7% of the samples were positive for both A. butzleri and A. cryaerophilus. A. skirrowii and A. cibarius were not detected in any of the chicken samples analyzed. The enrichment PCR assay developed is a specific and rapid alternative for the survey of Arcobacter contamination in meat.

scholarly journals The Likelihood of Misidentifying Rodent Pasteurellaceae by Using Results from a Single PCR Assay

2019 ◽  
Vol 58 (2) ◽  
pp. 201-207 ◽  
Author(s):  
Hagit Dafni ◽  
Lea Greenfeld ◽  
Roni Oren ◽  
Alon Harmelin
Keyword(s):  
Health Monitoring ◽  
Animal Health ◽  
16S Rrna Gene Sequencing ◽  
Pcr Analysis ◽  
Rrna Gene ◽  
Correct Identification ◽  
Pcr Assay ◽  
Specific Pcr ◽  
Primer Sets ◽  
Species Specific

The precise identification of rodent Pasteurellaceae is known to be highly challenging. An unknown strain of Pasteurellaceae appeared and rapidly spread throughout our animal facilities. Standard microbiology, combined with biochemical analysis, suggested that the bacteria strain was Rodentibacter pneumotropicus or R. heylii. We submitted samples of the unknown bacteria and known isolates of R. pneumotropicus, R. heylii, and Muribacter muris, to 2 service laboratories that provide animal health monitoring. Results of microbiology tests performed by both laboratories, species-specific PCR analysis performed by one laboratory, and independent 16S rRNA gene sequencing yielded identical identification of the unknown bacteria as Pasteurellaceae (Pasteurella spp.) and not R. pneumotropicus or R. heylii. In contrast, the similarly intended PCR assay performed by the other laboratory identified the bacteria as R. heylii. Careful evaluation of all of the results led us to conclude that the correct identification of the bacteria is Pasteurellaceae. From our experience, we recommend that a combination of several methods should be used to achieve correct identification of rodent Pasteurellaceae. Specifically, we advise that all primer sets used should be disclosed when reporting PCR test results, including in health reports provided by service laboratories and animal vendors. Careful, correct, and informative health monitoring reports are most beneficial to animal researchers and caretakers who might encounter the presence and effects of rodent Pasteurellaceae.

Arcobacter cibarius sp. nov., isolated from broiler carcasses

2005 ◽  
Vol 55 (2) ◽  
pp. 713-717 ◽  
Author(s):  
Kurt Houf ◽  
Stephen L. W. On ◽  
Tom Coenye ◽  
Jan Mast ◽  
Jan Van Hoof ◽  
...  
Keyword(s):  
Dna Hybridization ◽  
Type Strain ◽  
Pairwise Comparison ◽  
Rrna Gene ◽  
Biochemical Tests ◽  
Mean Values ◽  
Specific Pcr ◽  
Sds Page ◽  
Arcobacter Butzleri ◽  
Species Specific

Twenty Gram-negative, rod-shaped, slightly curved, non-spore-forming bacteria that gave a negative result in Arcobacter species-specific PCR tests but that yielded an amplicon in an Arcobacter genus-specific PCR test were isolated from 13 unrelated broiler carcasses. Numerical analysis of the profiles obtained by SDS-PAGE of whole-cell proteins clustered all isolates in a single group distinct from the other Arcobacter species. DNA–DNA hybridization among four representative strains exhibited DNA binding values above 91 %. DNA–DNA hybridization with reference strains of the current four Arcobacter species revealed binding levels below 47 %. The G+C contents ranged between 26·8 and 27·3 mol%. Pairwise comparison of 16S rRNA gene sequences revealed the mean values for similarity to the type strain of Arcobacter cryaerophilus (97·5 %), Arcobacter butzleri (96·5 %), Arcobacter skirrowii (96·0 %) and Arcobacter nitrofigilis (95·0 %). The levels of similarity to Campylobacter and Helicobacter species were below 88 and 87 %, respectively. The isolates could be distinguished from other Arcobacter species by the following biochemical tests: catalase, oxidase and urease activities; reduction of nitrate; growth at 25 and 37 °C under aerobic conditions; growth on 2–4 % (w/v) NaCl media; and susceptibility to cephalothin. These data demonstrate that the 20 isolates represent a single novel Arcobacter species, for which the name Arcobacter cibarius sp. nov. is proposed, with LMG 21996T (=CCUG 48482T) as the type strain.

scholarly journals Efficiency of PCR-RFLP and species-specific PCR for the identification of meat origin in dry sausages

2017 ◽  
Vol 35 (No. 5) ◽  
pp. 386-391
Author(s):  
Kušec Ivona Djurkin ◽  
Samac Danijela ◽  
Margeta Vladimir ◽  
Radišić Žarko ◽  
Vincek Dragutin ◽  
...  
Keyword(s):  
Rflp Analysis ◽  
Chicken Meat ◽  
Specific Primers ◽  
Specific Pcr ◽  
Sheep Meat ◽  
Pcr Rflp ◽  
Pork Sausages ◽  
Species Specific ◽  
Dry Sausages

The purpose of this investigation was the identification of chicken, beef and sheep meat in pork sausages using PCR-RFLP and PCR with pecies-specific primers. Six dry fermented pork sausages were produced by adding beef, sheep and chicken meat to each in the amount of 1 and 5%. DNA was extracted from five regions of each sausage and PCR-RFLP together with PCR using species-specific primers was performed. PCR-RFLP analysis was successful only for chicken meat, while species-specific PCR was effective for identification of chicken, eef and sheep meat in all ratios and from all regions of the sausages. The results of our study show that discovering adulteration using PCR-RFLP is suitable only for chicken meat in the investigated products, while for detection of beef and sheep meat use of species-specific oligonucleotides is more effective.

Development of a multiplex PCR assay for the detection of key genes associated with Pasteurella multocida subspecies

2021 ◽  
pp. 104063872110634
Author(s):  
Barbara Ujvári ◽  
Hubert Gantelet ◽  
Tibor Magyar
Keyword(s):  
Multiplex Pcr ◽  
Pasteurella Multocida ◽  
Rrna Gene ◽  
Pcr Assay ◽  
Biochemical Tests ◽  
Specific Primers ◽  
Multiplex Pcr Assay ◽  
Subspecies Level ◽  
Species Specific ◽  
Reference Strains

The ability to distinguish among the subspecies of Pasteurella multocida isolates is important epidemiologically; however, classification at the subspecies level based on the results of conventional biochemical tests (fermentation of sorbitol and dulcitol) is reportedly not accurate in all cases. Therefore, we developed a rapid, multiplex PCR assay to differentiate among the 3 subspecies of P. multocida. The PCR assay includes the P. multocida species–specific primers KMT1SP6 and KMT1T7 as an internal amplification control, with a newly designed gatD (galactitol-1-phosphate-5-dehydrogenase)-specific primer pair (unique for subsp. gallicida), and primers targeting a 16S rRNA gene region specific for subsp. septica. The subspecies specificity of the PCR was demonstrated by applying the test to a collection of 70 P. multocida isolates, including the Heddleston serovar reference strains; all isolates and strains were assigned correctly. The PCR assay is a sensitive, specific, and highly effective method for the identification of P. multocida subspecies, and an alternative to biochemical test–based differentiation. A possible relationship was noticed between P. multocida subspecies and lipopolysaccharide (LPS) genotype; all but one of the subsp. gallicida strains were isolated only from avian hosts and represented L1 LPS genotype. Subsp. multocida and subsp. septica isolates were classified into 5 and 4 different LPS genotypes, respectively, of which L3 was the only LPS genotype shared between these 2 subspecies.

Echinococcus multilocularis in a Eurasian lynx (Lynx lynx) in Turkey

Parasitology ◽  
2018 ◽  
Vol 145 (9) ◽  
pp. 1147-1150 ◽  
Author(s):  
Hamza Avcioglu ◽  
Esin Guven ◽  
Ibrahim Balkaya ◽  
Ridvan Kirman
Keyword(s):  
Echinococcus Multilocularis ◽  
Pcr Analysis ◽  
12S Rrna ◽  
Rrna Gene ◽  
Lynx Lynx ◽  
Eurasian Lynx ◽  
The North ◽  
Specific Pcr ◽  
Gene Sequence Analysis ◽  
Species Specific

AbstractEchinococcus multilocularis is the causative agent of alveolar echinococcosis (AE), one of the most threatening zoonoses in Eurasia. Human AE is widespread in the Erzurum region of Turkey, but the situation of the disease in intermediate and definitive hosts is unknown. A Eurasian lynx (Lynx lynx) was killed in a traffic accident in the north of Erzurum, and was taken to our laboratory. Sedimentation and counting technique (SCT), DNA isolation and polymerase chain reaction (PCR) analysis were performed. The SCT results showed that the lynx was infected with E. multilocularis with a medium (745 worms) worm burden. The DNA of adult worms obtained from the lynx was analyzed with a species-specific PCR, and the worms were confirmed to be E. multilocularis by 12S rRNA gene sequence analysis. This is the first report of E. multilocularis from Eurasian lynx in Turkey.

scholarly journals Distribution of Bifidobacterial Species in Human Intestinal Microflora Examined with 16S rRNA-Gene-Targeted Species-Specific Primers

1999 ◽  
Vol 65 (10) ◽  
pp. 4506-4512 ◽  
Author(s):  
Takahiro Matsuki ◽  
Koichi Watanabe ◽  
Ryuichiro Tanaka ◽  
Masafumi Fukuda ◽  
Hiroshi Oyaizu
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Intestinal Tract ◽  
Bifidobacterium Longum ◽  
Rrna Gene ◽  
Human Feces ◽  
Specific Primers ◽  
Specific Pcr ◽  
Species Specific ◽  
Human Intestinal Tract

ABSTRACT In order to clarify the distribution of bifidobacterial species in the human intestinal tract, a 16S rRNA-gene-targeted species-specific PCR technique was developed and used with DNAs extracted from fecal samples obtained from 48 healthy adults and 27 breast-fed infants. To cover all of the bifidobacterial species that have been isolated from and identified in the human intestinal tract, species-specific primers for Bifidobacterium longum, B. infantis,B. dentium, and B. gallicum were developed and used with primers for B. adolescentis, B. angulatum, B. bifidum, B. breve, and the B. catenulatum group (B. catenulatum andB. pseudocatenulatum) that were developed in a previous study (T. Matsuki, K. Watanabe, R. Tanaka, and H. Oyaizu, FEMS Microbiol. Lett. 167:113–121, 1998). The specificity of the nine primers was confirmed by PCR, and the species-specific PCR method was found to be a useful means for identifying Bifidobacteriumstrains isolated from human feces. The results of an examination of bifidobacterial species distribution showed that the B. catenulatum group was the most commonly found taxon (detected in 44 of 48 samples [92%]), followed by B. longum andB. adolescentis, in the adult intestinal bifidobacterial flora and that B. breve, B. infantis, andB. longum were frequently found in the intestinal tracts of infants. The present study demonstrated that qualitative detection of the bifidobacterial species present in human feces can be accomplished rapidly and accurately.

scholarly journals Species-Specific Plasmid Sequences for PCR Identification of the Three Species of Borrelia burgdorferiSensu Lato Involved in Lyme Disease

1998 ◽  
Vol 36 (1) ◽  
pp. 269-272 ◽  
Author(s):  
Marie-Christine Misonne ◽  
Philippe Pierre Hoet
Keyword(s):  
Lyme Disease ◽  
Pcr Amplification ◽  
Sensu Stricto ◽  
Pcr Assay ◽  
Borrelia Garinii ◽  
Specific Pcr ◽  
Pcr Identification ◽  
Identification Of Species ◽  
Species Specific ◽  
Specific Sequences

Species-specific sequences were shown to be carried by plasmids of the three main species of Borrelia burgdorferi sensu lato involved in Lyme disease. Libraries of the 16-, 33-, and 25-kb plasmids of B. burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii, respectively, were then built and used to isolate species-specific sequences. After sequencing of the cloned inserts, three sets of primers were designed. They were shown to determine species-specific PCR amplification products. The sensitivities of the PCR assay with these primers were 100 spirochetes for B. burgdorferi sensu stricto and 1,000 spirochetes for B. garinii and B. afzelii. The usefulness of these primers for the identification of species in biological samples (tick, serum, and cerebrospinal fluid samples) was ascertained.

scholarly journals Evaluation of Procedures for the Collection, Processing, and Analysis of Biomolecules from Low-Biomass Surfaces

2011 ◽  
Vol 77 (9) ◽  
pp. 2943-2953 ◽  
Author(s):  
K. Kwan ◽  
M. Cooper ◽  
M. T. La Duc ◽  
P. Vaishampayan ◽  
C. Stam ◽  
...  
Keyword(s):  
Molecular Analysis ◽  
Distinct Species ◽  
Pcr Analysis ◽  
Rrna Gene ◽  
Sample Collection ◽  
Sampling Device ◽  
Small Surface ◽  
Cellular Density ◽  
Specific Pcr ◽  
Species Specific

ABSTRACTTo comprehensively assess microbial diversity and abundance via molecular-analysis-based methods, procedures for sample collection, processing, and analysis were evaluated in depth. A model microbial community (MMC) of known composition, representative of a typical low-biomass surface sample, was used to examine the effects of variables in sampling matrices, target cell density/molecule concentration, and cryogenic storage on the overall efficacy of the sampling regimen. The MMC used in this study comprised 11 distinct species of bacterial, archaeal, and fungal lineages associated with either spacecraft or clean-room surfaces. A known cellular density of MMC was deposited onto stainless steel coupons, and after drying, a variety of sampling devices were used to recover cells and biomolecules. The biomolecules and cells/spores recovered from each collection device were assessed by cultivable and microscopic enumeration, and quantitative and species-specific PCR assays. rRNA gene-based quantitative PCR analysis showed that cotton swabs were superior to nylon-flocked swabs for sampling of small surface areas, and for larger surfaces, biological sampling kits significantly outperformed polyester wipes. Species-specific PCR revealed differential recovery of certain species dependent upon the sampling device employed. The results of this study empower current and future molecular-analysis-based microbial sampling and processing methodologies.

scholarly journals Species-specific RT-PCR amplification of human enteroviruses: a tool for rapid species identification of uncharacterized enteroviruses

2006 ◽  
Vol 87 (1) ◽  
pp. 119-128 ◽  
Author(s):  
M. Steven Oberste ◽  
Kaija Maher ◽  
Alford J. Williams ◽  
Naomi Dybdahl-Sissoko ◽  
Betty A. Brown ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
Pcr Primers ◽  
Human Enterovirus ◽  
Rt Pcr ◽  
Specific Primer ◽  
Specific Primers ◽  
Typical Application ◽  
Partial Sequencing ◽  
Specific Pcr ◽  
Species Specific

The 65 serotypes of human enteroviruses are classified into four species, Human enterovirus (HEV) A to D, based largely on phylogenetic relationships in multiple genome regions. The 3′-non-translated region of enteroviruses is highly conserved within a species but highly divergent between species. From this information, species-specific RT-PCR primers were developed that can be used to rapidly screen collections of enterovirus isolates to identify species of interest. The four primer pairs were 100 % specific when tested against enterovirus prototype strains and panels of isolates of known serotype (a total of 193 isolates). For evaluation in a typical application, the species-specific primers were used to screen 186 previously uncharacterized non-polio enterovirus isolates. The HEV-B primers amplified 68·3 % of isolates, while the HEV-A and HEV-C primers accounted for 9·7 and 11·3 % of isolates, respectively; no isolates were amplified with the HEV-D primers. Twelve isolates (6·5 %) were amplified by more than one primer set and eight isolates (4·3 %) were not amplified by any of the four primer pairs. Serotypes were identified by partial sequencing of the VP1 capsid gene, and in every case sequencing confirmed that the species-specific PCR result was correct; the isolates that were amplified by more than one species-specific primer pair were mixtures of two (11 isolates) or three (one isolate) species of viruses. The eight isolates that were not amplified by the species-specific primers comprised four new serotypes (EV76, EV89, EV90 and EV91) that appear to be unique members of HEV-A based on VP1, 3D and 3′-non-translated region sequences.

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