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.

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.

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 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.

Identification of non-tuberculous mycobacteria: utility of the GenoType Mycobacterium CM/AS assay compared with HPLC and 16S rRNA gene sequencing

2009 ◽  
Vol 58 (7) ◽  
pp. 900-904 ◽  
Author(s):  
Andie S. Lee ◽  
Peter Jelfs ◽  
Vitali Sintchenko ◽  
Gwendolyn L. Gilbert
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
16S Rrna Gene Sequencing ◽  
Rapid Identification ◽  
Mycobacterium Abscessus ◽  
Clinical Isolates ◽  
Rrna Gene ◽  
Specific Pcr ◽  
Clinically Significant ◽  
Species Specific

Non-tuberculous mycobacteria (NTM) causing clinical disease have become increasingly common and more diverse. A new reverse line probe assay, GenoType Mycobacterium CM/AS (Hain Lifescience), was evaluated for identification of a broad range of NTM. It was compared with phenotypic (HPLC) and molecular (DNA probes, in-house real-time multiplex species-specific PCR, 16S rRNA gene PCR and sequencing) identification techniques, which together provided the reference ‘gold standard’. A total of 131 clinical isolates belonging to 31 Mycobacterium species and 19 controls, including 5 non-Mycobacterium species, was used. Concordant results between the GenoType Mycobacterium assay and the reference identification were obtained in 119/131 clinical isolates (90.8 %). Identification of Mycobacterium abscessus and Mycobacterium lentiflavum by the assay was problematic. The GenoType Mycobacterium assay enables rapid identification of a broad range of potentially clinically significant Mycobacterium species, but some species require further testing to differentiate or confirm ambiguous results.

scholarly journals Species-specific PCR assay for the detection of Babesia odocoilei

2021 ◽  
pp. 104063872110329
Author(s):  
Hilary J. Burgess ◽  
Betty P. Lockerbie ◽  
Lisanework E. Ayalew ◽  
Antonia Dibernardo ◽  
Kristýna Hrazdilová ◽  
...  
Keyword(s):  
Dna Amplification ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Species Sensitivity ◽  
Pcr Assay ◽  
Banding Patterns ◽  
Specific Pcr ◽  
Pcr Product ◽  
Specific Pcr Assay ◽  
Species Specific

We developed a PCR assay for the detection of Babesia odocoilei based on the 18S rRNA gene. Multiple specimens of B. odocoilei were examined, and the assay consistently produced a small specific PCR product of 306 bp. The PCR assay was also challenged with DNA from 13 other Babesia species and 2 Theileria species, originating from 10 different host species; however, nonspecific DNA amplification and multiple banding patterns were observed, and the amplicon banding patterns varied between different isolates of the same species. Sensitivity was determined to be 6.4 pg of DNA, and an estimated 0.0001% parasitism. This assay can be utilized for species-specific differential detection of B. odocoilei.

scholarly journals Development and Validation of a Nested-PCR-Denaturing Gradient Gel Electrophoresis Method for Taxonomic Characterization of Bifidobacterial Communities

2003 ◽  
Vol 69 (11) ◽  
pp. 6380-6385 ◽  
Author(s):  
R. Temmerman ◽  
L. Masco ◽  
T. Vanhoutte ◽  
G. Huys ◽  
J. Swings
Keyword(s):  
Nested Pcr ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Temporal Changes ◽  
Rrna Gene ◽  
Specific Pcr ◽  
Gradient Gel Electrophoresis ◽  
Species Specific ◽  
Taxonomic Characterization

ABSTRACT The taxonomic characterization of a bacterial community is difficult to combine with the monitoring of its temporal changes. None of the currently available identification techniques are able to visualize a “complete” community, whereas techniques designed for analyzing bacterial ecosystems generally display limited or labor-intensive identification potential. This paper describes the optimization and validation of a nested-PCR-denaturing gradient gel electrophoresis (DGGE) approach for the species-specific analysis of bifidobacterial communities from any ecosystem. The method comprises a Bifidobacterium-specific PCR step, followed by purification of the amplicons that serve as template DNA in a second PCR step that amplifies the V3 and V6-V8 regions of the 16S rRNA gene. A mix of both amplicons is analyzed on a DGGE gel, after which the band positions are compared with a previously constructed database of reference strains. The method was validated through the analysis of four artificial mixtures, mimicking the possible bifidobacterial microbiota of the human and chicken intestine, a rumen, and the environment, and of two fecal samples. Except for the species Bifidobacterium coryneforme and B. indicum, all currently known bifidobacteria originating from various ecosystems can be identified in a highly reproducible manner. Because no further cloning and sequencing of the DGGE bands is necessary, this nested-PCR-DGGE technique can be completed within a 24-h span, allowing the species-specific monitoring of temporal changes in the bifidobacterial community.

scholarly journals Real-Time PCR Assay for Detection and Enumeration of Dekkera bruxellensis in Wine

2003 ◽  
Vol 69 (12) ◽  
pp. 7430-7434 ◽  
Author(s):  
Trevor G. Phister ◽  
David A. Mills
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pcr Primers ◽  
Rrna Gene ◽  
Dekkera Bruxellensis ◽  
Pcr Assay ◽  
Specific Pcr ◽  
Spoilage Yeast ◽  
Pcr Method ◽  
26S Rrna

ABSTRACT Traditional methods to detect the spoilage yeast Dekkera bruxellensis from wine involve lengthy enrichments. To overcome this difficulty, we developed a quantitative real-time PCR method to directly detect and enumerate D. bruxellensis in wine. Specific PCR primers to D. bruxellensis were designed to the 26S rRNA gene, and nontarget yeast and bacteria common to the winery environment were not amplified. The assay was linear over a range of cell concentrations (6 log units) and could detect as little as 1 cell per ml in wine. The addition of large amounts of nontarget yeasts did not impact the efficiency of the assay. This method will be helpful to identify possible routes of D. bruxellensis infection in winery environments. Moreover, the time involved in performing the assay (3 h) should enable winemakers to more quickly make wine processing decisions in order to reduce the threat of spoilage by D. bruxellensis.

scholarly journals Xylanolytic Psychotrophs From Andosolic Sedge Fens and Moss Heaths in Iceland

Fine Focus ◽  
2018 ◽  
Vol 4 (2) ◽  
pp. 171-186
Author(s):  
Olivia J. Rickman ◽  
M. Auður Sigurbjörnsdóttir ◽  
Oddur Vilhemsson
Keyword(s):  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Bacterial Strains ◽  
Cold Environments ◽  
Specific Pcr ◽  
Xylanolytic Activity ◽  
Cold Active ◽  
Rrna Gene Sequencing ◽  
Stenotrophomonas Rhizophila

Nine xylanolytic bacterial strains were isolated from fen and heath soils in northern Iceland. They were found by 16S rRNA gene sequencing to belong to the genera Paenibacillus, Bacillus, Pseudomonas, and Stenotrophomonas. Using a simple, plate-based semiquantitative assay with azo-crosslinked xylan as the substrate, it was determined that although isolated from cold environments, most of the strains displayed greater xylanolytic activity under mesophilic conditions, with only the paenibacilli displaying markedly cold-active xylanolytic activity. Indeed, for one isolate, Paenibacillus castaneae OV2122, xylanolytic activity was only detected at 15°C and below under the conditions tested. Of the nine strains, Paenibacillus amylolyticus OV2121 displayed the greatest activity at 5°C. Glycohydrolase family-specific PCR indicated that the paenibacilli produced multiple xylanases of families 10 and 11, whereas a family 8 xylanase was detected in Pseudomonas kilonensis AL1515, and a family 11 xylanase in Stenotrophomonas rhizophila AL1610.

scholarly journals REP-PCR Analysis to Study Prokaryotic Biodiversity from Lake Meyghan

2017 ◽  
Vol 61 ◽  
pp. 69-84 ◽  
Author(s):  
Ali Naghoni ◽  
Giti Emtiazi ◽  
Mohammad Ali Amoozegar ◽  
Zahra Etemadifar ◽  
Seyed Abolhassan Shahzadeh Fazeli
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Pcr Analysis ◽  
Rrna Gene ◽  
White Region ◽  
Rrna Gene Sequencing

Repetitive extragenic palindromic elements-polymerase chain reaction (rep-PCR) with 16S ribosomal ribonucleic acid (16S rRNA) genes sequences successfully used for the analysis of microbial community. In this study, the prokaryotic community in Lake Meyghan described by using rep-PCR analysis along with 16S rRNA gene sequencing. The water samples were collected from Lake Meyghan in November 2013. All samples were diluted and cultured on three different media. To estimate the number of prokaryotes per milliliter of the lake we used quantitative real‑time PCR (qPCR). Rep-PCR combination with 16S rRNA gene sequencing was performed to investigate prokaryotes biodiversity in the lake. 305 strains were isolated in this work; 113 isolates for green region, 102 isolates for red region, and 90 isolates for white region. The dendrograms generated 10, 7, and 9 clusters for a 70 % similarity cut-off for green, red, and white regions, respectively. Based on rep-PCR and 16S rRNA gene sequencing, the recovered isolates were dominated by (77.5 %)Halobacteriacaeand many isolates were related to the generaHalorubrum,Haloarcula,Haloterrigena,Natrinema, andHalovivaxin the white region. In the red region more isolated strains (57.5 %) belonged toBacillaceaeand the remaining 42.5 % of isolates belonged to archaea domain,Halorubrum, andHaloarcula. In the green region members ofGammaproteobacteriawere recoverd, this region was dominant withPseudoalteromonas,Salinivibrio, andAliidiomarina.

Development of an efficient PCR-based diagnosis protocol for the identification of the pinewood nematode, Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae)

Nematology ◽  
2004 ◽  
Vol 6 (2) ◽  
pp. 279-285 ◽  
Author(s):  
Jae Soon Kang ◽  
Kwang Sik Choi ◽  
Sang Chul Shin ◽  
Il Sung Moon ◽  
Sang Gil Lee ◽  
...  
Keyword(s):  
Intergenic Spacer ◽  
Nematode Species ◽  
Bursaphelenchus Xylophilus ◽  
5S Rrna ◽  
Rrna Gene ◽  
Pinewood Nematode ◽  
Pine Wood ◽  
5S Rrna Gene ◽  
Primer Sets ◽  
Species Specific

Abstract Pine wood wilt disease caused by the pine wood nematode, Bursaphelenchus xylophilus , has been a serious problem in the southern regions of Korea. Efficient diagnosis of B. xylophilus from infected pine wood specimens is critical for the management of this pest. Traditional microscopic examination often results in an erroneous identification because a closely related non-pathogenic species, B. mucronatus, has a great degree of morphological similarity to B. xylophilus. In an attempt to search for reliable molecular markers for the discrimination of these species, we have cloned the 5S rRNA genomic DNA fragments containing both coding and intergenic spacer (IGS) regions from B. xylophilus and B. mucronatus through a homology-probing PCR strategy. Sequence analyses revealed that coding sequences of the 5S rRNA gene from the two species are almost identical (98.3% homology) but that the IGS sequences differ substantially between the species. Based on the IGS sequence differences (69.7% homology), we designed species-specific primer sets and developed a PCR-based diagnosis protocol for the identification and discrimination of the two nematode species on a molecular basis.

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