A Novel Approach to the Viability Determination of Mycobacterium avium subsp. paratuberculosis Using Platinum Compounds in Combination With Quantitative PCR

Abstract

Mycobacterium avium subsp. paratuberculosis (MAP) represents a slow-growing bacterium causing paratuberculosis, especially in domestic and wild ruminants. Until recently, the assessment of MAP viability relied mainly on cultivation, which is very time consuming and is unable to detect viable but non-culturable cells. Subsequently, viability PCR, a method combining sample treatment with the DNA-modifying agent ethidium monoazide (EMA) or propidium monoazide (PMA) and quantitative PCR (qPCR), was developed, enabling the selective detection of MAP cells with an intact cell membrane. However, this technology requires a laborious procedure involving the need to work in the dark and on ice. In our study, a method based on a combination of platinum compound treatment and qPCR, which does not require such a demanding procedure, was investigated to determine mycobacterial cell viability. The conditions of platinum compound treatment were optimized for the fast-growing mycobacterium M. smegmatis using live and heat-killed cells. The optimal conditions consisting of a single treatment with 100 μM cis-dichlorodiammine platinum(II) for 60 min at 5°C resulted in a difference in quantification cycle (Cq) values between live and dead membrane-compromised mycobacterial cells of about 6 Cq corresponding to about 2 log10 units. This optimized viability assay was eventually applied to MAP cells and demonstrated a better ability to distinguish between live and heat-killed mycobacteria as compared to PMA. The viability assay combining the Pt treatment with qPCR thereby proved to be a promising method for the enumeration of viable MAP cells in foodstuffs, environmental, and clinical samples which could replace the time-consuming cultivation or laborious procedures required when using PMA.

Development of a Propidium Monoazide-Based Viability Quantitative PCR Assay for Red Sea Bream Iridovirus Detection

“…It has been reported that PtCl 4 penetrates living cells in small amounts and causes a slight decrease in the qPCR fluorescence signal. Furthermore, unlike PMAxx, it does not react with water molecules and becomes inactivated after reacting with viruses [ 33 ]. The suppression of qPCR after PtCl 4 treatment in this study could be because the PtCl 4 molecules were not eliminated and modified the DNA during the extraction procedure [ 33 ].…”

“…Furthermore, unlike PMAxx, it does not react with water molecules and becomes inactivated after reacting with viruses [ 33 ]. The suppression of qPCR after PtCl 4 treatment in this study could be because the PtCl 4 molecules were not eliminated and modified the DNA during the extraction procedure [ 33 ]. Our study of RSIV viability via qPCR using platinum compounds is valuable, considering that no such studies have been conducted previously.…”

An Assay Combining Droplet Digital PCR With Propidium Monoazide Treatment for the Accurate Detection of Live Cells of Vibrio vulnificus in Plasma Samples

“…The DNAs from dead cells could lead to an overestimation of live cell counts and produce a false-positive result, which is not conducive to clinical monitoring of V. vulnificus infection. Since the first study on using PMA to differentiate live cells from dead cells was reported in 2006 (Nocker et al, 2006), PMA has become one of the most widely used dyes (Codony et al, 2020) and been applied to many fields of microbiology (Deshmukh et al, 2020;Cechova et al, 2021;Hong et al, 2021). PMA could not bind to the DNA of live cells with the intact bacterial plasma membrane structure.…”

Optimization of the composition of a solid culture medium for Mycobacterium avium subsp. paratuberculosis using factorial design and response surface methodology

“…Immunological methods (such as blood or milk ELISA) lack sensitivity, particularly in the early stages of the disease (McAloon et al, 2020), and methods based on the detection of genetic material (such as PCR) are typically more sensitive but cannot differentiate between live and dead MAP cells (Plain et al, 2020). In order to demonstrate the viability of MAP detected by PCR, this method has been combined with viability dyes such as propidium monoazide (Cechova et al, 2021; Kralik et al, 2010), or bacteriophages as MAP lysing agents, since these only infect live bacteria (Foddai & Grant, 2020; Swift et al, 2020). Research continues to address the limitations of existing detection methods for MAP.…”