A new method for the rapid detection of the antibacterial and bacteriostatic activity of disinfectants based on Propidium Monoazide combined with real-time PCR

Abstract

Rapid detection of antibacterial and bacteriostatic properties is an important part of the quality and safety supervision of disinfectants. In this study, propidium monoazide (PMA) was used in combination with real-time PCR (PMA-qPCR) to detect the antibacterial and bacteriostatic activity of disinfectants against three commonly used indicator bacteria, Escherichia coli, Staphylococcus aureus, and Candida albicans, utilizing specifically designed primers. The method for preparing membrane-damaged bacteria was optimized to improve the ability of the PMA dye to distinguish between live and dead indicator bacteria. Finally, this method could simultaneously detect viable numbers of the indicator bacteria after the disinfectants were used. The R2 values of the PMA-qPCR standard curves were 0.9986, 0.9980, and 0.9962 for E. coli, S. aureus, and C. albicans, respectively, and the detection range was 103 ~ 106 CFU/ml, showing no significant difference in accuracy compared to that of the plate counting method (p > 0.05). The method established here is the first application of PMA-qPCR to detect the antibacterial and bacteriostatic activity of disinfectants. This technique markedly simplifies the detection steps of antibacterial and bacteriostatic activity, reduces the detection time (3 h compared to 48 ~ 72 h for the plate counting method), improves the quality supervision efficiency of disinfectants, and guarantees healthy and safe lives.

Efficacy of high‐level disinfection of endoscopes contaminated with Streptococcus equi subspecies equi with 2 different disinfectants

“…On the other hand, if restricted to the OPA, longer contact time (20 minutes) may help decrease the number of false positive qPCR results 7 . Finally, new updated qPCR assays are being used in human medicine and food safety that can distinguish between live and inactivated microbes (propidium monoazide combined qPCR tests) 24,25 . Propidium monoazide is a photoreactive dye that has a high affinity for DNA, and only penetrates damaged cell membranes 24,25 .…”

“…7 Finally, new updated qPCR assays are being used in human medicine and food safety that can distinguish between live and inactivated microbes (propidium monoazide qPCR tests). 24,25 Propidium monoazide is a photoreactive dye that has a high affinity for DNA, and only penetrates damaged cell membranes. 24,25 The ability to distinguish between live and dead organisms would be extremely useful in trying to determine if a horse (culture negative, but qPCR positive) is indeed a true carrier making residual DNA on endoscopes no longer a concern.…”

“… 7 Finally, new updated qPCR assays are being used in human medicine and food safety that can distinguish between live and inactivated microbes (propidium monoazide combined qPCR tests). 24 , 25 Propidium monoazide is a photoreactive dye that has a high affinity for DNA, and only penetrates damaged cell membranes. 24 , 25 The ability to distinguish between live and dead organisms would be extremely useful in trying to determine if a horse (culture negative, but qPCR positive) is indeed a true carrier making residual DNA on endoscopes no longer a concern.…”

Progress in methods for the detection of viable Escherichia coli

“…To date, researchers have dedicated considerable effort to devising swift and sensitive assays for identifying viable E. coli . Common markers used in PMA-PCR for detecting E. coli O157:H7 are Shiga toxin genes ( stx1 and stx2 ), 149 ycjM , 150,151 rfbE , 152 fliC , 153 wzy , 154 lacY , 155 ITS , 156 rRNAs, 157 lpfA and hlyA 158 (Table 2). Typically, these genes constitute virulence genes or shared phenotypic genes.…”

Fecal microbiome composition and diversity of cryopreserved canine stool at different duration and storage conditions

“…Traditional genomic DNA sequencing methods cannot distinguish DNA derived from bacteria with intact cell membranes from free DNA that leaked from bacteria with compromised cell membranes. Propidium monoazide (PMA) dye is a photoreactive chemical that preferentially binds double-stranded DNA, rendering it inert as a substrate for downstream PCR [ 6 ]. Propidium monoazide binds free DNA in solution and infiltrates bacteria with damaged cell membranes but cannot penetrate bacteria with intact cell membranes.…”