PCR Inhibition of a Quantitative PCR for Detection of Mycobacterium avium Subspecies Paratuberculosis DNA in Feces: Diagnostic Implications and Potential Solutions

Abstract

Molecular tests such as polymerase chain reaction (PCR) are increasingly being applied for the diagnosis of Johne's disease, a chronic intestinal infection of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). Feces, as the primary test sample, presents challenges in terms of effective DNA isolation, with potential for PCR inhibition and ultimately for reduced analytical and diagnostic sensitivity. However, limited evidence is available regarding the magnitude and diagnostic implications of PCR inhibition for the detection of MAP in feces. This study aimed to investigate the presence and diagnostic implications of PCR inhibition in a quantitative PCR assay for MAP (High-throughput Johne's test) to investigate the characteristics of samples prone to inhibition and to identify measures that can be taken to overcome this. In a study of fecal samples derived from a high prevalence, endemically infected cattle herd, 19.94% of fecal DNA extracts showed some evidence of inhibition. Relief of inhibition by a five-fold dilution of the DNA extract led to an average increase in quantification of DNA by 3.3-fold that consequently increased test sensitivity of the qPCR from 55 to 80% compared to fecal culture. DNA extracts with higher DNA and protein content had 19.33 and 10.94 times higher odds of showing inhibition, respectively. The results suggest that the current test protocol is sensitive for herd level diagnosis of Johne's disease but that test sensitivity and individual level diagnosis could be enhanced by relief of PCR inhibition, achieved by five-fold dilution of the DNA extract. Furthermore, qualitative and quantitative parameters derived from absorbance measures of DNA extracts could be useful for prediction of inhibitory fecal samples.

Keywords: Johne’s disease (JD) diagnosis; Mycobacterium paratuberculosis; PCR inhibition; fecal PCR; qPCR diagnosis; relief PCR inhibition.

FIGURE 1

A diagrammatic representation of the experiments. A total of 296 archived DNA extract and the respective fecal sample were used. The result of HT-J qPCR performed on the archival DNA was used to select corresponding 125 fecal samples. Corresponding fecal culture results were also accessed post HT-J qPCR.

FIGURE 2

(A) Scatter plot of log₁₀ DNA quantified by HT-J qPCR of undiluted fecal DNA extracts against diluted DNA extracts from Experiment 1. The solid black line is the ‘line of equality.’ (B) Bland–Altman difference plot comparing log₁₀ DNA quantity of the undiluted and diluted and fecal DNA extracts from Experiment 1, by plotting the difference in MAP DNA quantified against the average DNA quantified. The solid blue line represents the optimal agreement also termed the ‘zero line’ and the region of agreement (95% confidence interval) is plotted as black lines. The dotted red line shows the average difference and the solid green line shows the regression line of the difference on the average. (C) Standard curve plotted by combining results from four individual standard curves constructed using 0.001–10 pg per reaction of MAP genomic DNA concentration. The point represents the mean Ct and the error bars represent ±SD of Ct.

FIGURE 3

Receiver operating curve (ROC curve) for HT-J qPCR test constructed using test results of 296 fecal samples comprising of 80 fecal culture positive and 216 fecal culture negative samples. The area under the curve (AUC) was 0.852 (95% CI: 0.7934, 0.9108).

FIGURE 4

Box and whisker plot showing absorbance ratios (A) A₂₆₀/A₂₈₀, (B) A₂₆₀/A₂₃₀, and (C) A₂₆₀/A₂₇₀ by inhibition status of the DNA extracts. (A) Shows a significant difference (p < 0.001) in the A₂₆₀/A₂₈₀ ratios between inhibitory and non-inhibitory samples. Likewise, (C) shows a significant difference in rank for absorbance ratios A₂₆₀/A₂₇₀ of inhibitory and non-inhibitory samples in a Mann–Whitney test (p < 0.001). ^∗Rrepresents significant difference.

FIGURE 5

Quantity of DNA and protein by inhibition status of the DNA extracts. Box-and whisker plots showing (A). quantity of DNA (ng/μl) and (B). quantity of protein (mg/ml) of DNA extracts in Experiment 2 by their inhibition status. The geometric means of both the DNA and protein quantities between the inhibitory and non-inhibitory DNA extracts were significantly different by two sample t-test (p < 0.0001). ^∗Represents significant difference.