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.

Regional fresh snowfall microbiology and chemistry are driven by geography in storm-tracked events, Colorado, USA

“…Though we caution too much extrapolation from the presence of just a few genera, we think it is fair to suggest that demonstrated differences between Sunshine and Golden are the result of environmental/physical differences between the two geographic sites—and may implicate urban/rural lines in anthropogenic bioaerosol-sourcing. In a seasonal analysis of precipitation (predominately liquid, only one snow sample) in the Greater Tokyo Area, Japan, Hiraoka et al (2017) report similar abundant phyla within their samples (e.g., Proteobacteria , Bacteroidetes , and Firmicutes —but more Actinobacteria than we report). Interestingly, Hiraoka et al saw few differences between sampling sites relative to the geographic differences that we see.…”

“…In vivo studies of both atmospheric (suspended as an aerosol) and hail (within the hailstone) microorganisms have shown them to be metabolically active ( Sattler, Puxbaum & Psenner, 2001 ; Ariya et al, 2002 ; Amato et al, 2007a , 2007c ; Temkiv et al, 2012 ), suggesting that the biomass in the environment from which snowfall derives is not of inconsequence and that microbiota can remain metabolically active throughout the rain/ice/snow nucleation process. Save for some work on liquid precipitation ( Hiraoka et al, 2017 ), what microbiota within ice/snow look like when they contact soil and surface receiving waters remains unknown. Beyond the community structure of precipitation microbiota, it is especially unknown how these structures changes as a result of geography and atmospheric events.…”

Culture-Independent Identification of Mycobacterium avium Subspecies paratuberculosis in Ovine Tissues: Comparison with Bacterial Culture and Histopathological Lesions

“…Conversely, samples not detected by qPCR, due to low MAP numbers in the tissues, sampling site tested or PCR inhibition, might be detected by culture. PCR inhibition and remediation strategies have been previously reported when assessing fecal sample types ( 50 ). Although no obvious evidence of inhibition was found in this study, based on the assessment of the quality of DNA extracts and testing and relief of potential PCR inhibition ( 50 ) by performing the qPCR test of fivefold diluted DNA extracts (data not shown), the absence of PCR inhibition cannot be guaranteed as it is a complex phenomenon.…”

Control of Mycobacterium avium subsp. paratuberculosis infection on a New Zealand pastoral dairy farm

“…While detection of the organism via fecal culture on Herrold’s egg yolk medium has been a definitive test for MAP infection this requires prolonged incubation periods of up to 16 weeks and may be compromised by overgrowth by contaminating gut organisms [13–15]. Internationally, the rapid, direct and quantitative measurement of MAP shedding in feces of infected and affected animals by quantitative PCR is rapidly becoming the standard and widely used method for JD diagnostic testing [16–20].…”