Advances in the nucleic acid detection method integrated with propidium monoazide to determine the viral infectivity / 中华实验和临床病毒学杂志

Currently, a number of analytical method, such as animal infection test, cell culture assay and polymerase chain reaction, are available for virus detection in clinical samples. The nucleic acid detection method has the highest sensitivity and specificity, but it was not established that the correlation between the detected viral genome and viral infectivity. The nucleic acid detection method integrated with propidium monoazide (PMA) can remedy this shortcoming to determine the viral infectivity. This review firstly describes the principle and procedure of the nucleic acid detection method integrated with PMA. It secondly analyzes the advantages and limitations of the nucleic acid detection method integrated with PMA. Finally, it discusses the influence of the viral structure, the inactivation mode and experimental conditions on the nucleic acid detection method integrated with PMA and summarise the progress of the method.

Development of a propidium monoazide-polymerase chain reaction assay for detection of viable Lactobacillus brevis in beer

ABSTRACT The spoilage of beer by bacteria is of great concern to the brewer as this can lead to turbidity and abnormal flavors. The polymerase chain reaction (PCR) method for detection of beer-spoilage bacteria is highly specific and provides results much faster than traditional microbiology techniques. However, one of the drawbacks is the inability to differentiate between live and dead cells. In this paper, the combination of propidium monoazide (PMA) pretreatment and conventional PCR had been described. The established PMA-PCR identified beer spoilage Lactobacillus brevis based not on their identity, but on the presence of horA gene which we show to be highly correlated with the ability of beer spoilage LAB to grow in beer. The results suggested that the use of 30 µg/mL or less of PMA did not inhibit the PCR amplification of DNA derived from viable L. brevis cells. The minimum amount of PMA to completely inhibit the PCR amplification of DNA derived from dead L. brevis cells was 2.0 µg/mL. The detection limit of PMA-PCR assay described here was found to be 10 colony forming units (CFU)/reaction for the horA gene. Moreover, the horA-specific PMA-PCR assays were subjected to 18 reference isolates, representing 100% specificity with no false positive amplification observed. Overall the use of horA-specific PMA-PCR allows for a substantial reduction in the time required for detection of potential beer spoilage L. brevis and efficiently differentiates between viable and nonviable cells.

Bactericidal effect of nano-silver against E.faecalis growing in multi-species biofilm / 实用口腔医学杂志

Objective:To evaluate the bactericidal effects of nano-silver against Enterococcus faecalis(E.faecalis) growing in multi-species biofilm.Methods:85 biofilms were established using MBECTM P&G Assay with E.faecalis together with Fusobacterium nucleatum and P.melaninogenica.Thereafter,10 specimens were used as the controls,75 were randomly divided into 3 groups(n=25),and treated with 0.1% nano-silver (12-15 nm) solution,0.1% nano-silver (100 nm) solution and 2% hypochlorite solution,respectively.Each sample was then separated into 2 different tubes.PMA was added to one of the tubes,and the other was left untreated.Then,DNA extraction and qPCR were performed.The cycle threshold(Ct) values between samples were compared by paired t test.Results:The Ct values of the samples treated with PMA were higher than that without PMA(P=0.000) in the group of 0.1% nano-silver solution(12-15 nm)was higher than that in the group of 0.1% nano-silver solution(100 nm)and 2% sodium hypochlorite solution.(P<0.05);the value in the group of 0.1% nano-silver solution(100 nm)was larger than that in 2% sodium hypochlorite solution(P<0.05).Conclusion:0.1% nano silver solution might have a strong bactericidal effect against against E.faecalis growing in multi-species biofilm.The bactericidal effect may be enhanced with the small size of silver particles.

Evaluation of Propidium Monoazide Real-Time PCR for Viablity of Mycobacterium leprae / 나학회지

BACKGROUND: Conventional acid-fast bacilli (AFB) staining cannot differentiate viable from dead cells. Propidium monoazide (PMA) is a photoreactive DNA-binding dye that inhibits PCR amplification by DNA modification. OBJECT: The author evaluated whether PMA real-time PCR is suitable for the viablity of Mycobacterium leprae (M. leprae) in specimens of cultivation in mouse foot pads. METHODS: A total of 55 diluted suspensions from mouse foot pads were quadruplicated and subjected to PMA treatment and/or heat inactivation, and were also tested to compare the ΔCT values (CT value in PMA-treated samples-CT value in non-PMA-treated samples). Real-time PCR was performed using QuantiTect SYBR® Green PCR Kits(Qiagen, USA), and the CT value changes after PMA treatment were compared between PMA treatment and/or heat inactivation groups. RESULTS: The increase in the CT value after PMA treatment was significant in heat inactivated group(4.26) and non-heat inactivated group(1.12)(both P = 0.000). In the ROC curve analysis, the cutoff ΔCT value for maximum sensitivity (100%) and specificity (97.1%) for differentiating dead from live cells was 2.41 CONCLUSIONS: PMA real-time PCR is a useful approach for evaluating viablity of M. leprae.

Quantitative evaluation of live Enterococcus faecalis in failed endodontic root-well-filled teeth / 实用口腔医学杂志

Objective:To analyse viable Enterococcus faecalis(E.faecalis)in root-well-filled teeth associated with failed endodontic treatment by using propidium monoazide (PMA)in combination with real time qPCR.Methods:Bacterial samples were extracted from 34 root-canal-treated teeth with post-treatment apical periodontitis.Each sample was separated into 2 different tubes.PMA was added to one of the tubes,and the other was left untreated.Then,DNA extraction and qPCR were performed.Results:E.faecalis was found in 20 of the 34 samples(58.8%).In PMA treated and none-treaten samples the Ct value of E.faecalis was 25.1 2 ±2.04 and 24.62 ± 2.02 respectively(P =0.001 ).Conclusion:PMA may be feasible in differentiating viable and dead Enterococcus faecalis cells.

Evaluation of Propidium Monoazide Real-Time PCR for Early Detection of Viable Mycobacterium tuberculosis in Clinical Respiratory Specimens

BACKGROUND: Conventional acid-fast bacilli (AFB) staining cannot differentiate viable from dead cells. Propidium monoazide (PMA) is a photoreactive DNA-binding dye that inhibits PCR amplification by DNA modification. We evaluated whether PMA real-time PCR is suitable for the early detection of viable Mycobacterium tuberculosis (MTB) in clinical respiratory specimens. METHODS: A total of 15 diluted suspensions from 5 clinical MTB isolates were quadruplicated and subjected to PMA treatment and/or heat inactivation. Eighty-three AFB-positive sputum samples were also tested to compare the DeltaC(T) values (C(T) value in PMA-treated sputum samples-C(T) value in non-PMA-treated sputum samples) between culture-positive and culture-negative specimens. Real-time PCR was performed using Anyplex MTB/NTM Real-Time Detection (Seegene, Korea), and the C(T) value changes after PMA treatment were compared between culture-positive and culture-negative groups. RESULTS: In MTB suspensions, the increase in the C(T) value after PMA treatment was significant in dead cells (P=0.0001) but not in live cells (P=0.1070). In 14 culture-negative sputum samples, the median DeltaC(T) value was 5.3 (95% confidence interval [CI], 4.1-8.2; P<0.0001), whereas that in 69 culture-positive sputum samples was 1.1 (95% CI, 0.7-2.0). In the ROC curve analysis, the cutoff DeltaC(T) value for maximum sensitivity (89.9%) and specificity (85.7%) for differentiating dead from live cells was 3.4. CONCLUSIONS: PMA real-time PCR is a useful approach for differentiating dead from live bacilli in AFB smear-positive sputum samples.

Selective detection of viable Enterococcus faecalis using propidium monoazide in combination with real-time PCR / 대한치과보존학회지

Polymerase chain reaction (PCR) can detect bacteria more rapidly than conventional plate counting. However DNA-based assays cannot distinguish between viable and dead cells due to persistence of DNA after cells have lost their vitality. Recently, propidium monoazide (PMA) treatment has been introduced. The purpose of this study is to evaluate the applicability of the PMA treatment and real-time PCR method for cell counting in comparison with plate counting and to evaluate the antibacterial efficacy of 2% CHX on E. faecalis using PMA treatment in combination with real-time PCR. Firstly, to elucidate the relationship between the proportion of viable cells and the real-time PCR signals after PMA treatment, mixtures with different ratios of viable and dead cells were used. Secondly, relative difference of viable cells using PMA treatment in combination with real-time PCR was compared with CFU by plate counting. Lastly, antibacterial efficacy of 2% CHX on E. faecalis was measured using PMA treatment in combination with real-time PCR. The results were as follows : 1. Ct value increased with decreasing proportion of viable E. faecalis. 2. There was correlation between viable cells measured by real-time PCR after PMA treatment and CFU by plate counting until Optical density (OD) value remains under 1.0. However, viable cells measured by real-time PCR after PMA treatment have decreased at 1.5 of OD value while CFU kept increasing. 3. Relative difference of viable E. faecalis decreased more after longer application of 2% CHX.