Development and Validation of a Quantitative Real-Time PCR Assay for The Molecular Diagnosis of Johne's Disease in Dairy Cattle

Abstract

Paratuberculosis (Johne’s disease) imposes a significant problem to the world dairy and beef industries; today it is considered as a potential zoonosis disease.This disease is caused by Mycobacterium avium subsp. paratuberculosis and it is characterized by progressive weight loss and profuse diarrhoea. So, the aim of thisstudy is to evaluate the fast and sensitive protocol between conventional PCR by using two primer sets (IS900 and F57) and real-time PCR using IS900 primer for diagnosis of Johne's disease in dairy cattle, in addition to review its effect on milk production of the same dairy cattle.This by collecting 150 samples (faecal and milk) from apparently healthy and suspect animals from different dairy farms in Egypt, the samples were grouped according to the status of the animal healthy into diarrhetic and non-diarrhetic samples. All this samples were tested by conventional using IS900 and F57 primers and by real-time PCR using IS900 primer set. The results indicated that using real-time in the diagnosis is more sensitive than conventional PCR since the percentage was 42% by real-time and 31.33% by conventional PCR in faecal samples, while the sensitivity of the two primer was nearly the same since the percentage of F57 was 28.67% there were no significant difference, but the milk samples had shown no significant different between the two techniques and the two primer sets, since the positive percentage of IS900 real-time, IS900 conventional PCR and F57 conventional PCR were 5.3%, 4.67% and 3.33%, respectively. The results were the same on the pure DNA, the real-time PCR was more sensitive than conventional PCR but the F57 primer was more sensitive than IS900 primer. When the samples were grouped according to the healthy status we found that in diseased animal there was no significant difference between the two techniques and the two primer sets but in the apparently healthy animals the sensitivity of real-time was more than conventional PCR but there was no significant difference between the two primers (IS900 and F57). And regarding the governorates, El-Behera embodies the highest rank of infected animals and El-Dakahlia was the lowest infected area.