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PCR Test for the Detection of Mycobacterium avium subsp. Paratuberculosis


Código: ADI045-100. Categorías: , , .

1. Purpose of the test
ADIAVET™ PARATB REAL TIME kit is intended to detect Mycobacterium avium subsp paratuberculosis
(PARATB) using real-time Polymerase Chain Reaction (PCR) technology from faeces, tissue and milk
specimens of bovine, ovine and caprine, as well as from environmental specimen and bacterial culture.
2. Pathogen
Mycobacterium avium subsp. paratuberculosis is the etiological agent of bovine paratuberculosis. The
disease is characterized by diarrhoeas, a decrease of the production level (milk, reproduction) and a
loss of weight leading to death.
In 1898, Johne and Frothingham detect acid-fast bacillus in intestinal mucosa of affected animals.
These bacteria, similar to tuberculosis bacillus, are responsible for a thickening of intestinal mucosa
corresponding to an enteropathy called paratuberculosis or Johne’s disease (Thorel and al. , 1990). The
incubation of illness is slow (between 2 and 5 years) so the majority of affected animals present clinical
symptoms between 2 and 7 years. The most important mode of transmission of paratuberculosis is the
faecal-oral route, although transmission can occur in utero, via infected semen, colustrum and milk. In
the infected animal organisms, the bacteria can then spread through the macrophages.
Affected animals can shed varying numbers of M. paratuberculosis organisms in their faeces (from some
bacteria / g of faecal material to 104- 1010germs / g at the clinical stage).
Feacal culture for the causative organisms is the definitive method of diagnosis but it is very slow,
requiring 6-8 weeks. Immunologically-based tests for Johne’s disease are rapid but lack of specificity
and sensitivity.
A shift of biologists of St George’s Hospital of London conducted by the Dr J. Hermon-Taylor identified
in 1985 a repetitive genomic fragment called IS900, specific of M. paratuberculosis strains (Green and
al. 1990). Since this sequence has been used as probe in molecular diagnostic test in particular for PCR
test (Guillou and al., 1993).
3. Description and purpose of the test
This test is based on enzymatic gene amplification or PCR technology.
Amplified products are detected in real-time thanks to a specific labelled hydrolysis probe (5’-
exonulease technology).
The ADIAVET™ PARATB REAL TIME kit enables the simultaneous detection of:
– Mycobacterium avium subsp paratuberculosis (probe labelled in FAM),
– External Control (probe labelled with a fluorochrome with the same spectra as VIC and
HEX ).
According to the extraction protocol retained, two external controls are available:
– An External Positive Control of extraction named “EPC–Ext”, it will be added during the
extraction step, will follow all the step of extraction and will check the whole extraction
process and the absence of inhibitors.
– An External Positive Control of amplification named “EPC–Amp”; it will be added in the
“A5 solution “ before the amplification step and will only control the absence of
amplification inhibition.
ADIAGENE recommends DNA purification kits coming from ADIAGENE, Qiagen, Macherey-Nagel
suppliers. Other purification kits can be used if they have been validated by the user.

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