scholarly journals Detection of Mycobacterium avium Subspecies Paratuberculosis in Pooled Fecal Samples by Fecal Culture and Real-Time PCR in Relation to Bacterial Density

Animals ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1605
Author(s):  
Annika Wichert ◽  
Esra Einax ◽  
Natalie Hahn ◽  
Anne Klassen ◽  
Karsten Donat
Keyword(s):  
Real Time ◽  
Mycobacterium Avium ◽  
Detection Probability ◽  
Mycobacterium Avium Subspecies Paratuberculosis ◽  
Positive Association ◽  
Significant Loss ◽  
Positive Sample ◽  
Bacterial Density ◽  
Fecal Samples ◽  
Real Time Quantitative Pcr

Within paratuberculosis control programs Mycobacterium avium subsp. paratuberculosis (MAP)-infected herds have to be detected with minimum effort but with sufficient reliability. We aimed to evaluate a combination of random sampling (RS) and pooling for the detection of MAP-infected herds, simulating repeated RS in imitated dairy herds (within-herd prevalence 1.0%, 2.0%, 4.3%). Each RS consisted of taking 80 out of 300 pretested fecal samples, and five or ten samples were repeatedly and randomly pooled. All pools containing at least one MAP-positive sample were analyzed by culture and real-time quantitative PCR (qPCR). The pool detection probability was 47.0% or 45.9% for pools of size 5 or 10 applying qPCR and slightly lower using culture. Combining these methods increased the pool detection probability. A positive association between bacterial density in pools and pool detection probability was identified by logistic regression. The herd-level detection probability ranged from 67.3% to 84.8% for pools of size 10 analyzed by both qPCR and culture. Pools of size 10 can be used without significant loss of sensitivity compared with pools of size 5. Analyzing randomly sampled and pooled fecal samples allows the detection of MAP-infected herds, but is not recommended for one-time testing in low prevalence herds.

scholarly journals Identification, Detection, and Enumeration of Human Bifidobacterium Species by PCR Targeting the Transaldolase Gene

2002 ◽  
Vol 68 (5) ◽  
pp. 2420-2427 ◽  
Author(s):  
Teresa Requena ◽  
Jeremy Burton ◽  
Takahiro Matsuki ◽  
Karen Munro ◽  
Mary Alice Simon ◽  
...  
Keyword(s):  
Real Time ◽  
16S Rdna ◽  
Quantitative Pcr ◽  
Gene Sequence ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Bifidobacterium Longum ◽  
Bifidobacterium Adolescentis ◽  
Fecal Samples ◽  
Real Time Quantitative Pcr ◽  
Pcr Targeting

ABSTRACT Methods that enabled the identification, detection, and enumeration of Bifidobacterium species by PCR targeting the transaldolase gene were tested. Bifidobacterial species isolated from the feces of human adults and babies were identified by PCR amplification of a 301-bp transaldolase gene sequence and comparison of the relative migrations of the DNA fragments in denaturing gradient gel electrophoresis (DGGE). Two subtypes of Bifidobacterium longum, five subtypes of Bifidobacterium adolescentis, and two subtypes of Bifidobacterium pseudocatenulatum could be differentiated using PCR-DGGE. Bifidobacterium angulatum and B. catenulatum type cultures could not be differentiated from each other. Bifidobacterial species were also detected directly in fecal samples by this combination of PCR and DGGE. The number of species detected was less than that detected by PCR using species-specific primers targeting 16S ribosomal DNA (rDNA). Real-time quantitative PCR targeting a 110-bp transaldolase gene sequence was used to enumerate bifidobacteria in fecal samples. Real-time quantitative PCR measurements of bifidobacteria in fecal samples from adults correlated well with results obtained by culture when either a 16S rDNA sequence or the transaldolase gene sequence was targeted. In the case of samples from infants, 16S rDNA-targeted PCR was superior to PCR targeting the transaldolase gene for the quantification of bifidobacterial populations.

Evaluation of a Commercial Real-Time PCR Kit for the Detection of Mycobacterium avium subspecies paratuberculosis in Milk

2016 ◽  
Vol 73 (5) ◽  
pp. 668-675 ◽  
Author(s):  
Ahmad Alajmi ◽  
Günter Klein ◽  
Nils. Th. Grabowski ◽  
Svenja Fohler ◽  
Ömer Akineden ◽  
...  
Keyword(s):  
Real Time ◽  
Mycobacterium Avium ◽  
Real Time Pcr ◽  
Mycobacterium Avium Subspecies Paratuberculosis

scholarly journals Antibiotic Resistance Genes in the Bacteriophage DNA Fraction of Human Fecal Samples

2013 ◽  
Vol 58 (1) ◽  
pp. 606-609 ◽  
Author(s):  
Pablo Quirós ◽  
Marta Colomer-Lluch ◽  
Alexandre Martínez-Castillo ◽  
Elisenda Miró ◽  
Marc Argente ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Real Time ◽  
Quantitative Pcr ◽  
Resistance Genes ◽  
Antibiotic Resistance Genes ◽  
Fecal Samples ◽  
Real Time Quantitative Pcr ◽  
Healthy Humans ◽  
Phage Dna

ABSTRACTA group of antibiotic resistance genes (ARGs) (blaTEM,blaCTX-M-1,mecA,armA,qnrA, andqnrS) were analyzed by real-time quantitative PCR (qPCR) in bacteriophage DNA isolated from feces from 80 healthy humans. Seventy-seven percent of the samples were positive in phage DNA for one or more ARGs.blaTEM,qnrA, and,blaCTX-M-1were the most abundant, andarmA,qnrS, andmecAwere less prevalent. Free bacteriophages carrying ARGs may contribute to the mobilization of ARGs in intra- and extraintestinal environments.

Real-Time Quantitative PCR Detection of Mycobacterium avium Subspecies in Meat Products

2011 ◽  
Vol 74 (4) ◽  
pp. 636-640 ◽  
Author(s):  
B. KLANICOVA ◽  
I. SLANA ◽  
H. VONDRUSKOVA ◽  
M. KAEVSKA ◽  
I. PAVLIK
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Mycobacterium Avium ◽  
General Public ◽  
Meat Products ◽  
Mycobacterium Avium Subsp ◽  
Insertion Sequences ◽  
Real Time Quantitative Pcr ◽  
Qpcr Method ◽  
Meat Samples

The aim of this work was to examine various purchased meat products and to find out if any traces of Mycobacterium avium subsp. avium, M. avium subsp. hominissuis, and M. avium subsp. paratuberculosis could be detected in these samples. Analysis of the meat products (raw, cooked, and fermented) was performed using a real-time quantitative PCR (qPCR) method for the detection of specific insertion sequences: duplex qPCR for the detection of IS900 specific for M. avium subsp. paratuberculosis, and triplex qPCR for the detection of IS901 specific for Mycobacterium avium subsp. avium and IS1245 specific for M. avium subsp. hominissuis. Of the 77 analyzed meat samples, 17 (22%) were found to contain M. avium subsp. paratuberculosis DNA, 4 (5%) samples contained Mycobacterium avium subsp. avium DNA, and in 12 (16%) samples M. avium subsp. hominissuis DNA was detected. The concentration of M. avium subsp. paratuberculosis and M. avium subsp. hominissuis DNA in some meat products exceeded 104 genomes per g. Culture examination of these mycobacterial subspecies was negative. By analyzing a range of meat products, we have provided evidence to support the hypothesis that M. avium is present in everyday commodities sold to the general public

scholarly journals Comparison and Sensitivity Evaluation of Three Different Commercial Real-Time Quantitative PCR Kits for SARS-CoV-2 Detection

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1321
Author(s):  
Ana Banko ◽  
Gordana Petrovic ◽  
Danijela Miljanovic ◽  
Ana Loncar ◽  
Marija Vukcevic ◽  
...  
Keyword(s):  
Real Time ◽  
Positive Association ◽  
N Gene ◽  
Nucleic Acid Detection ◽  
Real Time Quantitative Pcr ◽  
Sensitivity Evaluation ◽  
Strong Positive Association ◽  
Primer Sets ◽  
Analytical Sensitivities

Real-time reverse transcription polymerase chain reaction (RT-qPCR) is the most sensitive and specific assay and, therefore, is the “gold standard” diagnostic method for the diagnosis of SARS-CoV-2 infection. The aim of this study was to compare and analyze the detection performance of three different commercially available SARS-CoV-2 nucleic acid detection kits: Sansure Biotech, GeneFinderTM, and TaqPathTM on 354 randomly selected samples from hospitalized COVID-19 patients. All PCR reactions were performed using the same RNA isolates and one real-time PCR machine. The final result of the three evaluated kits was not statistically different (p = 0.107), and also had a strong positive association and high Cohen’s κ coefficient. In contrast, the average Ct values that refer to the ORF1ab and N gene amplification were significantly different (p < 0.001 and p < 0.001, respectively), with the lowest obtained by the TaqPathTM for the ORF1ab and by the Sansure Biotech for the N gene. The results show a high similarity in the analytical sensitivities for SARS-CoV-2 detection, which indicates that the diagnostic accuracy of the three assays is comparable. However, the SanSure Biotech kit showed a bit better diagnostic performance. Our findings suggest that the imperative for improvement should address the determination of cut-off Ct values and rapid modification of the primer sets along with the appearance of new variants.

SEROPREVALENCE AND MOLECULAR IDENTIFICATION OF MYCOBACTERIUM AVIUM SUBSPECIES PARATUBERCULOSIS IN FARMED GOATS IN CENTRAL-SOUTHERN TAIWAN

2018 ◽  
Vol 44 (04) ◽  
pp. 189-196
Author(s):  
Heng-Ching Lin ◽  
Wai How Chong ◽  
Han Hsiang Huang ◽  
Chi-Chung Chou ◽  
Yi-Lun Tsai ◽  
...  
Keyword(s):  
Molecular Identification ◽  
Mycobacterium Avium ◽  
Mycobacterium Avium Subspecies Paratuberculosis ◽  
Seroprevalence Rate ◽  
Serum Samples ◽  
Fecal Samples ◽  
Identification Rate ◽  
Individual Level ◽  
Seroprevalence Data ◽  
Southern Taiwan

Mycobacterium avium subspecies paratuberculosis (MAP) are Gram positive, aerobic, acid-fast, catalase positive bacteria. The Johne’s disease, caused by MAP, is a wasting disease found in all ruminants including cattle, sheep, goats, deer, camelids and wildlife ruminants. MAP has attracted hygienic attention due to the link between Crohn’s disease (CD) in humans and MAP presence in the gut of patients. The aims of this study are to investigate and monitor the serological prevalence and molecular identification rate of MAP in caprine feces and verify the MAP-negative goat farms in central-southern Taiwan. A total of 8486 blood samples were randomly collected between the years 2011 and 2015 from 321 caprine farms. The serum samples were assessed by commercial ELISA while 3015 fecal samples from 201 anti-MAP antibodies (MAP-Ab) negative herds were further molecularly examined by polymerase chain reaction (PCR) from year 2014 to 2015. The individual seroprevalence rate of caprine MAP in 2011, 2012, 2013, 2014 and 2015 was 0% (0/1032), 0% (0/429), 0% (0/1402), 0.14% (4/2917) and 0.07% (2/2706), respectively. Molecular identification rate of MAP in caprine fecal samples at MAP-Ab negative farms accounted for 0.92% (14/1515) and 0.93 (14/1500), respectively, in the years 2014 and 2015. Meanwhile, there was no association between the MAP seroprevalence and the sampling regions or years at farm or individual level. The seroprevalence data revealed in this study highlighted the rising prevalence of caprine MAP and the link of MAP to farmed ruminant species and its possible implications in hygienic aspects.

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