Real-time quantitative PCR detection of Mycobacterium avium subsp. paratuberculosis and differentiation from other mycobacteria using SYBR Green and TaqMan assays

Ravva, Subbarao V.; Stanker, Larry H.

doi:10.1016/j.mimet.2005.04.004

Cited by 41 publications

(31 citation statements)

References 35 publications

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“…3 In the current study, additional procedures were incorporated into the traditional guanidine lysis method to make it more suitable for the purification of MAP DNA from bovine fecal samples. First, NaOH pretreatment facilitated the liberation of MAP organisms bound within fecal matter and/or contaminated soil materials, thereby improving the process of organism recovery 19 ; the step also helped to eliminate PCR inhibitors as reported previously. 20 The combination of mechanical disruption and 1-tube chemical lysis/extraction allowed for maximal recovery of MAP DNA and reduction of cross-contamination.…”

Section: Discussionmentioning

confidence: 89%

“…To culture MAP organisms from feces, specimens were processed as previously described. 19 In brief, 1 gram of feces was thoroughly mixed with 35 ml of doubledistilled water for 30 min and allowed to stand undisturbed at room temperature for 30 min. The supernatant (30 ml) was transferred to a new tube and centrifuged at 1,800 3 g for 30 min.…”

Section: Bacterial Strain and Growth Conditionsmentioning

confidence: 99%

“…19 In brief, the reaction mixture consisted of DNase-and RNase-free water, 13 TaqMan Universal PCR Master Mix, k 300 nM primers, 75 nM probe, and 5 ml of template DNA. The PCR primers and probe were commercially synthesized, l and the PCR assays were performed on a sequence detection system m under the following conditions: 1 cycle of 94uC for 5 min; 45 cycles of 95uC for 15 sec and 60uC for 1 min.…”

Section: Real-time Pcr Assaysmentioning

confidence: 99%

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An Efficient DNA Extraction Method for Polymerase Chain Reaction–Based Detection of Mycobacterium Avium Subspecies Paratuberculosis in Bovine Fecal Samples

Zhang

2011

J VET Diagn Invest

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Abstract. Due to the lipid rich cell wall of Mycobacterium avium subspecies paratuberculosis (MAP), the complex nature of bovine feces, and intermittent organism shedding by infected cattle, it is difficult to recover a sufficient amount of high-quality MAP DNA from fecal samples, directly affecting the sensitivity of downstream polymerase chain reaction (PCR) tests. In the current study, a DNA extraction method, designated the Mississippi Veterinary Research and Diagnostic Laboratory (MVRDL) method, was developed for PCR-based detection of MAP in bovine fecal samples. The MVRDL method combined multiple procedures, including chemical pretreatment, 1-tube cell lysis and extraction, chelex matrix absorption, and mini-column purification. The DNA yield and purity, as measured by spectrophotometry, was 3.36 fg per colony forming unit (CFU) MAP and A260/280 absorbance ratio of 2, respectively. This method was further evaluated by real-time PCR. A linear correlation was found between cycle-threshold (Ct) and log input CFU (ranging from 7.2 to 7.2 3 10 7 CFU per ml or CFU per g). The detection limit of the realtime PCR assay was 3 CFU per ml of MAP culture or per g of MAP-spiked feces. In addition, the MVRDL method was validated by performing 7 Johne's direct fecal PCR proficiency tests administered by the National Veterinary Service Laboratories. Based on culture results as the ''gold standard,'' the specificity of MVRDL PCR was 100%, and the sensitivity was 98.46% for samples containing more than 1.5 CFU per tube of fecal cultures. To the authors' knowledge, this is the most efficient MAP DNA extraction method in comparison with all previously published protocols.

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Section: Discussionmentioning

confidence: 89%

Section: Bacterial Strain and Growth Conditionsmentioning

confidence: 99%

Section: Real-time Pcr Assaysmentioning

confidence: 99%

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An Efficient DNA Extraction Method for Polymerase Chain Reaction–Based Detection of Mycobacterium Avium Subspecies Paratuberculosis in Bovine Fecal Samples

Zhang

2011

J VET Diagn Invest

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“…The primer sequences were SF214; 5'-ATGACGGTTACGGAG-GTGGTT-3' (forward primer), SR289; 5'-TGCAGTAATG-GTCGGCCTTAC-3' (reverse primer), and PR265; 5'-FAM CGACCACGCCCGCCCAGATAMRA-3' (probe) as described previously [11]. The real-time PCR reaction was conducted using a Rotor-Gene Q real-time PCR cycler (Qiagen, USA) and a reaction mixture consisting of 1× Rotor-Gene Probe PCR master mix (Qiagen), 400 nM primers, 100 nM probe, 4 µL of template DNA, and nuclease-free water to give a total volume of 20 µL.…”

mentioning

confidence: 99%

Effective DNA extraction method to improve detection of Mycobacterium avium subsp. paratuberculosis in bovine feces

Park¹,

Shin²,

Sung³

et al. 2014

Korean Journal of Veterinary Research

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: Paratuberculosis caused by Mycobacterium avium subsp. paratuberculosis (MAP) has extended latent periods of infection. Due to this property, difficulties in the detection of fecal shedder have been raised. A newly designed method for DNA extraction from fecal specimens, mGITC/SC was evaluated in terms of diagnostic efficiency. The detection limit of IS900 real-time PCR was about 50 MAP (1.5 cfu) in 250 mg of feces (6 cfu per g). Also, this DNA extraction method was faster and cheaper than that using commercial kit or other methods. Consequently, the mGITC/ SC is an economical DNA extraction method that could be a useful tool for detecting MAP from fecal specimens.

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“…Over the last few years, new molecular methods have been introduced, including PCR-restriction fragment length polymorphism (RFLP), real-time PCR, DNA sequencing, and DNA strip technology, leading to a considerable improvement in both the speed and accuracy of mycobacterial identification (10). All methods have advantages and limitations; in general, those with a high specificity and a low minimum detection limit are expensive and complex to perform (2,11,12,14). The development of a sensitive and specific diagnostic assay that can be applied directly to clinical samples without the need for high-cost dedicated equipment will definitely improve diagnostic investigation of mycobacterial infections.…”

mentioning

confidence: 99%

Specific Detection of Unamplified Mycobacterial DNA by Use of Fluorescent Semiconductor Quantum Dots and Magnetic Beads

et al. 2010

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Here we present the development of a specific DNA detection method using fluorescent semiconductor quantum dots (QDs) and magnetic beads (MBs) for fast detection of Mycobacterium spp., dispensing with the need for DNA amplification. Two biotinylated oligonucleotide probes were used to recognize and detect specific complementary mycobacterial target DNA through a sandwich hybridization reaction. Cadmium selenite QDs conjugated with streptavidin and species-specific probes were used to produce a fluorescent signal. MBs conjugated with streptavidin and a genus-specific probe were used to isolate and concentrate the DNA targets. The application of the proposed method to isolated bacteria produced the expected result in all cases. The minimum detection limit of the assay was defined as 12.5 ng of DNA diluted in a sample volume of 20 l. In order to obtain an indication of the method's performance with clinical samples, we applied the optimized assay to the detection of Mycobacterium tuberculosis in DNA isolated from bronchoalveolar lavage specimens from patients with tuberculosis and Mycobacterium avium subsp. paratuberculosis in DNA isolated from feces and paraffin-embedded tissues in comparison with culture, Ziehl-Neelsen staining, and real-time PCR. The concordance of these methods compared to the proposed method with regard to positive and negative samples varied between 53.84% and 87.23% and between 84.61% and 100%, respectively. The overall accuracy of the QD assay compared to real-time PCR was 70 to 90% depending on the type of clinical material. The proposed diagnostic assay offers a simple, rapid, specific, and cost-effective method for direct detection and identification of mycobacterial DNA in clinical samples.

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Real-time quantitative PCR detection of Mycobacterium avium subsp. paratuberculosis and differentiation from other mycobacteria using SYBR Green and TaqMan assays

Cited by 41 publications

References 35 publications

An Efficient DNA Extraction Method for Polymerase Chain Reaction–Based Detection of Mycobacterium Avium Subspecies Paratuberculosis in Bovine Fecal Samples

An Efficient DNA Extraction Method for Polymerase Chain Reaction–Based Detection of Mycobacterium Avium Subspecies Paratuberculosis in Bovine Fecal Samples

Effective DNA extraction method to improve detection of Mycobacterium avium subsp. paratuberculosis in bovine feces

Specific Detection of Unamplified Mycobacterial DNA by Use of Fluorescent Semiconductor Quantum Dots and Magnetic Beads

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