Mycobacterium Paratuberculosis DNA Test Kit, Polymerase Chain Reaction
Detection of M. paratuberculosis DNA
- detects to 1 gene copy of DNA
Frequently Asked Questions (FAQs)
Q. What is the principle of the real-time PCR test?
A. The test is based on polymerase chain reaction (PCR) that, in addition to specific forward and reverse oligonucleotide primers, utilizes fluorogenic probe hydrolysis chemistry to generate a fluorescent signal when specific M. paratuberculosis DNA is present in samples. The primers and probe target the hspX gene, which contains specific DNA sequences that allow for molecular discrimination of M. paratuberculosis from other closely related Mycobacterium species.
Q. Should all samples which give a Ct value before the end-point be considered positive, even the really late ones? Do you recommend cut-off values for positive samples?
A. We recommend that a sample be considered positive if:
- Ct < 38 for ABI and BioRad series of real-time PCR thermocyclers
- Ct < 42 for Cepheid and Roche real-time PCR thermocyclers
Samples which produce Ct values in these ranges can with high confidence be considered positive. These recommended cut-off values are at the upper range of the Ct values at which one gene copy of DNA can be detected (in an uninhibited sample) on the designated thermocyclers.
A sample that appears to cross the threshold after the cutoff Ct value can not definitely be considered positive (“suspect” result). Additional testing would be required to confirm.
Q. Can I pool fecal samples?
A. Pooling of fecal samples can be both a time-saving and cost-effective means for determining herd infection status. Fecal pools can be of two types: (1) artificially pooled by tester or (2) naturally pooled as co-mingled manure from environmental samples. Our MAP Extraction System and real-time PCR test (Mycobacterium Paratuberculosis DNA Test Kit, Polymerase Chain Reaction) can be used with both types of pooled fecal samples. For artificially pooled samples, we currently recommend pools of equal amounts of fecal samples up to 5 cows/pool (e.g., 2 gm fecal sample from each cow mixed together). At this level, the presence of 1 or more super-shedders, heavy shedders, or moderate shedders per pool will be easily detected. In addition, in most cases, the presence of 1 or more low shedders should also be detected in the pool.
Q. Is there any time I should not pool fecal samples?
A. For determining and monitoring infection status in a commercial herd, pooling of samples is a cost-effective means. However, testing of individual fecal samples is recommended for seed-stock or replacement cattle.
Q. Can I use this kit to help diagnosis Johne’s disease in other animal species?
A. This kit should be used for testing bovine fecal samples or for confirmation of cultures derived from bovine samples. No claims are made for other samples or for organisms derived from other animal species.
Q. Can I use frozen fecal samples?
A. Fresh, moist fecal samples will give the best results and correlate best with cfu counts from HEYM culture. Fecal samples which were promptly frozen and continuously stored at -80oC are acceptable; however, repeated freezing and thawing of fecal samples should be avoided, as DNA may degrade. [NOTE: Dry fecal samples can produce sub-optimal results and should be avoided].
For technical questions, contact Tracy:
Johne's Technical Representative
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