Optimization and Validation of a Diagnostic Real-Time PCR for Specific Detection of Mycobacterium avium Subspecies Paratuberculosis

Abstract

| A diagnostic Real time PCR (qPCR) for detection of Mycobacterium avium subspecies paratuberculosis (MAP) was optimized using the ISMav2 sequence specific for MAP. The analytical sensitivity of the assay was 500 fg, and it was able to detect approximately 7 copies of the positive control plasmid construct. The qPCR detected up to 1x10 3 , 1x10 4 and 1x 10 5 MAP cells per reaction from spiked bovine semen, milk and feces respectively. The assay was reliable, reproducible and could be completed in 87 minutes. Comparative quantification for MAP copy number was established by utilizing normalized Cq values. The ISMav2 qPCR specifically detected 8 MAP strains, but was unable to amplify DNA from any other strains of Mycobacterium (n=5) or non-Mycobacterium strains (n=10). Several factors were tested to study their impact on the validation of the assay under field conditions. It was observed that sample size, number of sampling time points and test methods adopted for accepting the infection status were strongly correlated. The best return of validation estimates were obtained when sample-wise results of the qPCR were compared to the combined status of culture and acid fast bacilli (AFB) staining. The diagnostic sensitivity and specificity, positive and negative predictive values were estimated to be 100% (95% CI |63.06 -100.0|) and 99.9% (95% CI |99.44 100.0|), 88.89% (CI |51.75 -99.72|) and 100% (95%CI |99.75 -100.0|), respectively. Also, the qPCR and the MAP identification test conditions were strongly associated (κ = 0.941 (95% CI |0.825 -1.00|). The validation estimates were true only when the sample size was large (n=1008) and sample collection was based on a single time point sampling strategy. Thus it appears that the assay could be considered as a reliable and rapid diagnostic test for field application. Keywords

Integrative analysis of metabolome and transcriptome profiles provides insight into the fruit pericarp pigmentation disorder caused by ‘Candidatus Liberibacter asiaticus’ infection

“…The primers of 16 S rDNA were P1(5-’GCGCGTATGCAATACGAGCGGC-3’)/P2(5’-GCCTCGCGACTTCGCAACCCAT-3’). The recombinant plasmid pMD-18T-16 S rDNA was used for constructing a standard curve and equation relating the plasmid copy number and CT value [ 30 ]. According to the equation, the HLB bacterial population of Candidatus Liberibacter asiaticus in pericarps was calculated.…”

“…According to the equation, the HLB bacterial population of Candidatus Liberibacter asiaticus in pericarps was calculated. The plasmid copy number was calculated based on the equation ‘(amount in ng× 6.022 × 10 23 )/(length in bp×1 × 10 9 ng/g ×650 g/mole of bp) [ 30 ], where length = 3829 bp’. The amplification system consisted of 11.2 µL of probe qRT-PCR premix system (Takara, Ishiyama, Japan), 0.4 µL each of 10 µmol/L forward and reverse primer, 2 µL of cDNA template, and sterile distilled water to a total volume of 20 µL, and the protocol was 95 ℃ for 20 s followed by 40 cycles at 95 ℃ for 1 s and 60 ℃ for 20 s in a StepOnePlus Real-Time PCR System (Applied Biosystems, USA).…”