A semi-automated magnetic capture probe based DNA extraction and real-time PCR method applied in the Swedish surveillance of Echinococcus multilocularis in red fox (Vulpes vulpes) faecal samples

Campylobacter jejuni infection is a significant cause of foodborne gastroenteritis worldwide. Consumption and handling of poultry products is believed to be the primary risk factor for campylobacteriosis. Risk assessments require quantitative data, and C. jejuni is enumerated usually by direct plating, which sometimes allows growth of non-Campylobacter bacteria. The objective of the present study was to develop a quantitative real-time PCR method (q-PCR) for enumerating C. jejuni in chicken rinse without a culturing step. The procedure to obtain the template for the PCR assay involved (i) filtration of 10 ml of chicken rinse, (ii) centrifugation of the sample, and (iii) total DNA extraction from the pellet obtained using a commercial DNA extraction kit. The detection limit of the method was comparable to that for plating 100 μl of chicken rinse on modified charcoal cefoperazone deoxycholate agar, and the detection limit could be further improved 10-fold by concentrating the DNA eluate by ethanol precipitation. A close correlation for spiked chicken rinse was obtained for the results of the quantitative real-time PCR method and direct plating (r = 0.99). The coefficient of correlation for the methods was 0.87 when samples from chicken carcasses on the slaughter line were analyzed, whereas a lower correlation (r = 0.76) was obtained when samples from retail carcasses were analyzed. Greater variation in the proportion of dead and/or viable but not culturable Campylobacter types in the retail samples may explain the decreased correlation between the methods. Overall, the new method is simple and fast and the results obtained are closely correlated with those for direct plating for samples containing a low proportion of dead Campylobacter cells.

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Detection of Rendered Meat and Bone Meals by PCR Is Dependent on Animal Species of Origin and DNA Extraction Method

Journal of Food Protection ◽

10.4315/0362-028x-73.6.1090 ◽

2010 ◽

Vol 73 (6) ◽

pp. 1090-1096 ◽

Cited By ~ 9

Author(s):

MICHAEL J. MYERS ◽

DOROTHY E. FARRELL ◽

CHRISTINE M. DEAVER ◽

JACQULINE MASON ◽

HEIDI L. SWAIM ◽

...

Keyword(s):

Real Time ◽

Dna Extraction ◽

Real Time Pcr ◽

Animal Species ◽

Animal Feed ◽

Functional Assay ◽

Threshold Values ◽

Meat And Bone Meal ◽

Bone Meal ◽

Pcr Method

The capability of eight commercially available DNA extraction kits to extract bovine DNA originating in meat and bone meal from fortified feed was evaluated. Four different batches of bovine meat and bone meal (BMBM) were used for DNA extraction with the eight commercial DNA extraction kits. Within each kit, there were minimal differences in the batch-to-batch amounts of extracted DNA. There were differences between the kits in the amounts of DNA that could be extracted from the same amount of starting BMBM. These differences did not translate into differences in the amount of amplifiable DNA from BMBM-fortified dairy feed. Using a validated real-time PCR method, the kit yielding the highest amount extractable DNA was completely unable to yield a positive PCR result; one other kit was also unable to produce a positive PCR result from DNA extracted from BMBM-fortified feed. There was a complete lack of a correlation between the amount of bovine DNA isolated from BMBM by a given extraction kit compared with the relative amounts of DNA isolated from fortified animal feed as evidenced by the cycle threshold values generated using the real-time PCR method. These results demonstrate that extraction of DNA from processed animal protein is different for pure ingredients and fortified animal feeds. These results indicate that a method specifically developed using just animal-derived meat and bone meal may not yield a functional assay when used to detect animal tissues in complete animal feed.

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A more sensitive, efficient and ISO 17025 validated Magnetic Capture real time PCR method for the detection of archetypal Toxoplasma gondii strains in meat

International Journal for Parasitology ◽

10.1016/j.ijpara.2017.05.005 ◽

2017 ◽

Vol 47 (13) ◽

pp. 875-884 ◽

Cited By ~ 9

Author(s):

Ignacio Gisbert Algaba ◽

Manon Geerts ◽

Malgorzata Jennes ◽

Wim Coucke ◽

Marieke Opsteegh ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Real Time ◽

Real Time Pcr ◽

Iso 17025 ◽

Magnetic Capture ◽

Pcr Method

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Real time PCR to detect the environmental faecal contamination by Echinococcus multilocularis from red fox stools

Veterinary Parasitology ◽

10.1016/j.vetpar.2013.12.023 ◽

2014 ◽

Vol 201 (1-2) ◽

pp. 40-47 ◽

Cited By ~ 41

Author(s):

Jenny Knapp ◽

Laurence Millon ◽

Lorane Mouzon ◽

Gérald Umhang ◽

Francis Raoul ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Echinococcus Multilocularis ◽

Red Fox ◽

Faecal Contamination

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Development of a Magnetic Capture Hybridization Real-Time PCR Assay for Detection of Tumorigenic Agrobacterium vitis in Grapevines

Phytopathology ◽

10.1094/phyto-10-12-0267-r ◽

2013 ◽

Vol 103 (6) ◽

pp. 633-640 ◽

Cited By ~ 12

Author(s):

Kameka L. Johnson ◽

Desen Zheng ◽

Supaporn Kaewnum ◽

Cheryl Lynn Reid ◽

Thomas Burr

Keyword(s):

Real Time ◽

Dna Extraction ◽

Real Time Pcr ◽

Detection Threshold ◽

Pcr Primers ◽

Immunomagnetic Separation ◽

Extraction Methods ◽

Economic Effects ◽

Agrobacterium Vitis ◽

Magnetic Capture

Agrobacterium vitis, the causal agent of grape crown gall, can have severe economic effects on grape production. The bacterium survives systemically in vines and, therefore, is disseminated in propagation material. We developed an assay for use in indexing programs that is efficient and sensitive for detecting A. vitis in grape tissue. Initially, real-time polymerase chain reaction (PCR) primers specific for diverse tumorigenic strains of A. vitis were developed using the virD2 gene sequence. To overcome the effects of PCR inhibitors present in plant tissue, DNA extraction methods that included magnetic capture hybridization (MCH), immunomagnetic separation (IMS), and extraction with the Mo Bio Powerfood kit were compared. The assays incorporating MCH or IMS followed by real-time PCR were 10,000-fold more sensitive than direct real-time PCR when tested using boiled bacterial cell suspensions, with detection thresholds of 101 CFU/ml compared with 105 CFU/ml. DNA extraction with the Powerfood DNA extraction kit was 10-fold more sensitive than direct real-time PCR, with a detection threshold of 104 CFU/ml. All three assays were able to detect A. vitis in healthy-appearing grapevine cuttings taken from infected vines.

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Comparison of real-time PCR and the Kato-Katz method for the diagnosis of soil-transmitted helminthiasis and assessment of cure in a randomized controlled trial

BMC Microbiology ◽

10.1186/s12866-020-01963-9 ◽

2020 ◽

Vol 20 (1) ◽

Cited By ~ 1

Author(s):

Beatrice Barda ◽

Christian Schindler ◽

Rahel Wampfler ◽

Shaali Ame ◽

Said M. Ali ◽

...

Keyword(s):

Real Time ◽

Dna Extraction ◽

Real Time Pcr ◽

Controlled Trial ◽

Bead Beating ◽

Novel Treatments ◽

Two Samples ◽

Stool Samples ◽

Pcr Method

Abstract Background Diagnosis of soil-transmitted helminths (STHs) in developing countries is commonly based on microscopic detection of eggs in stool samples, using the Kato-Katz (KK) method, which has a poor sensitivity for detecting light intensity infections. We compared the performance of the KK method and real-time PCR in the framework of a randomized trial, which evaluated four novel treatments against Trichuris trichiura and concomitant STH infections. Results Two stool samples obtained from 320 participants were examined at baseline and follow-up with quadruplicate KK and PCR analyses of one of the two samples using “bead-beating” for DNA extraction. At follow-up, 80 samples were negative according to both PCR and KK and 173 were positive with both methods for any of the STHs. Relative to PCR, the calculated sensitivity of KK at follow-up was 83.6%, 43.0% and 53.8% for T. trichiura, for hookworm and for Ascaris lumbricoides, respectively. The sensitivity of PCR compared with KK at this time point was 89.1% for T. trichiura, 72.7% for hookworm and 87.5% for A. lumbricoides. Cure rates (CRs) for T. trichiura and A. lumbricoides were slightly lower with the PCR method. For hookworm CRs with KK were mostly significantly lower, namely 36.7%, 91.1%, 72.2% and 77.8% for moxidectin, moxidectin in combination with tribendimidine, moxidectin in combination with albendazole and albendazole in combination with oxantel pamoate, respectively, whereas with PCR the CRs were 8.3%, 82.6%, 37.1% and 57.1%, respectively. Conclusions In conclusion, a single real-time PCR is as sensitive as quadruplicate KK for T. trichiura and A. lumbricoides detection but more sensitive for hookworm, which has an influence on the estimated treatment efficacy. PCR method with DNA extraction using the “bead-beating protocol” should be further promoted in endemic areas and laboratories that can afford the needed equipment. The study is registered at ISRCTN (no. 20398469).

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