Development of propidium monoazide combined with real-time quantitative PCR (PMA-qPCR) assays to quantify viable dominant microorganisms responsible for the traditional brewing of Hong Qu glutinous rice wine

Lv, Xu-Cong; Li, Yan; Qiu, Wanwei; Wu, Xiaoqi; Xu, Bei; Liang, Yiting; Liu, Bin; Chen, Shaojun; Rao, Pingfan; Ni, Li

doi:10.1016/j.foodcont.2016.01.040

Cited by 39 publications

(17 citation statements)

References 38 publications

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“…They have been widely applied to quantitatively detect the early onset of diseases with window periods such as virus, oncogenes, etc. Furthermore, DNA dyes such as EMA (ethidium monoazide bromide azide) and PMA (propidium monoazide bromide azide) have been extensively used to distinguish nucleic acids of live and dead cells [63,64]. These dyes can penetrate damaged cells and bind to DNA with little effect on live cells endowed from their intact cell membranes [65,66].…”

Section: Fluorescent Dye-based Analysismentioning

confidence: 99%

Strand Displacement Amplification for Multiplex Detection of Nucleic Acids

Zeng¹,

Mukama²,

Lu³

et al. 2019

Modulating Gene Expression - Abridging the RNAi and CRISPR-Cas9 Technologies

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The identification of various targets such as bacteria, viruses, and other cells remains a prerequisite for point-of-care diagnostics and biotechnological applications. Nucleic acids, as encoding information for all forms of life, are excellent biomarkers for detecting pathogens, hereditary diseases, and cancers. To date, many techniques have been developed to detect nucleic acids. However, most of them are based on polymerase chain reaction (PCR) technology. These methods are sensitive and robust, but they require expensive instruments and trained personnel. DNA strand displacement amplification is carried out under isothermal conditions and therefore does not need expensive instruments. It is simple, fast, sensitive, specific, and inexpensive. In this chapter, we introduce the principles, methods, and updated applications of DNA strand displacement technology in the detection of infectious diseases. We also discuss how robust, sensitive, and specific nucleic acid detection could be obtained when combined with the novel CRISPR/Cas system.

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Section: Fluorescent Dye-based Analysismentioning

confidence: 99%

Strand Displacement Amplification for Multiplex Detection of Nucleic Acids

Zeng¹,

Mukama²,

Lu³

et al. 2019

Modulating Gene Expression - Abridging the RNAi and CRISPR-Cas9 Technologies

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“…It is necessary to investigate the effect of non-viable bacteria on the analysis of the microbial community by DNA-based molecular technologies. Non-viable microbes were reported to affect the estimation of microbial community diversity in meconium (Stinson et al, 2019), clinical feces (Young et al, 2017), topsoil (Carini et al, 2017), rice wine (Lv et al, 2016), and cheese (Erkus et al, 2016), but not that in the samples of groundwater (Lopez-Fernandez et al, 2018) or soil battery (Gustave et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

“…Given that nonviable microbes may be generated during CSFL production, it is necessary to evaluate the influence of non-viable microbes on the accurate characterization of the PM microbial community. Propidium monoazide (PMA), as a kind of DNA molecular dye, can enter non-viable microbes and interact with their DNA to inhibit DNA amplification (Lv et al, 2016). But PMA cannot enter live microbe cells and thus cannot affect their DNA molecules (Lv et al, 2016).…”

Section: Introductionmentioning

confidence: 99%

“…Propidium monoazide (PMA), as a kind of DNA molecular dye, can enter non-viable microbes and interact with their DNA to inhibit DNA amplification (Lv et al, 2016). But PMA cannot enter live microbe cells and thus cannot affect their DNA molecules (Lv et al, 2016). By integrating PMA into conventional quantitative polymerase chain reaction (qPCR) and 16S rRNA gene high-throughput sequencing, we were able to remove non-viable bacteria from PM samples and then compare the differences in copy number, community diversity, and composition between the viable bacteria and total bacteria.…”

Section: Introductionmentioning

confidence: 99%

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Detection of Viable and Total Bacterial Community in the Pit Mud of Chinese Strong-Flavor Liquor Using Propidium Monoazide Combined With Quantitative PCR and 16S rRNA Gene Sequencing

et al. 2020

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Microbiota in the pit mud (PM) plays a crucial role in the production of Chinese strongflavor liquor (CSFL), the most popular distilled liquor in China. However, previous studies used total microbes, instead of viable ones, for the characterization of the microbial community in this environment. In this study, we used propidium monoazide (PMA) combined with quantitative polymerase chain reaction (qPCR) and 16S rRNA gene sequencing to verify the effect of non-viablee bacteria on the characterization of PM bacteria. After PMA concentration optimization, 50 µM PMA was chosen to pretreat 5 and 20 years PMs. The qPCR results showed that there were 50.78 and 71.84% of non-viable bacteria in the 5-year PM and 20-year PM, respectively. Both copy numbers of total bacteria and viable bacteria were significantly higher in 20-year PM than those in 5-year PM. Nevertheless, in terms of bacterial diversity and composition analyses at the operational taxonomic unit (OTU), phylum, class, and genus levels, 16S rRNA gene sequencing results displayed no significant differences between total bacteria and viable bacteria in both PM types. In conclusion, it is necessary for non-viable bacteria to be considered in determining absolute biomass of bacteria in PM, but not necessary in the analysis of diversity and composition of PM bacteria. To the best of our knowledge, our study is the first attempt to analyze viable bacteria in the PM of CSFL and provides useful information on how to accurately characterize a microbial community in a PM environment.

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“…Moreover, certain cases showed persistent DNA positivity within several months. However, this scientific significance was hampered by the limitations of their study as the method could not tell the DNA of viable M. pneumoniae apart from nonviable ones [20]. us, methods that can accurately measure the amount of M. pneumoniae in throat are required to evaluate the clinical efficiency of treatment options.…”

Section: Introductionmentioning

confidence: 99%

Real-Time PCR and Quantitative Culture for Mycoplasma pneumoniae Load in Pharyngeal Swabs from Children at Preliminary Diagnosis and Discharge

Zhao

Guan

et al. 2020

BioMed Research International

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Background. Extensive studies have focused on the diagnosis and treatment of Mycoplasma pneumoniae infection; however, rare studies investigated the posttreatment conditions. We analyzed the carrying status of M. pneumoniae in the respiratory tract of children before and after treatment. Methods. Ninety-two children with M. pneumoniae pneumonia were included in this study. Clinical data were obtained from each patient, and pharyngeal swab sampling was performed at preliminary diagnosis and discharge. Real-time PCR and dilution quantitative culture were utilized to determine the DNA quantification and number of viable M. pneumoniae from samples collected upon preliminary diagnosis and discharge. Results. All the 92 cases showed DNA positivity upon preliminary diagnosis, serum IgM antibody was detected in 80 patients, and positivity of M. pneumoniae culture was observed in 82 cases. Upon discharge, the M. pneumoniae nucleotide and culture positivity were detected in 87 and 49 cases, respectively. The content of viable M. pneumoniae was 10–104 CCU/mL and 10–102 CCU/mL in the preliminary diagnosis samples and discharge samples, respectively. Conclusions. Real-time PCR was rapid and effective for the qualitative diagnosis of M. pneumoniae at the early stage, but it cannot be used to evaluate the prognosis of patients with M. pneumoniae infection. Quantitative analysis for M. pneumoniae DNA could not directly reflex the viable strain content.

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Development of propidium monoazide combined with real-time quantitative PCR (PMA-qPCR) assays to quantify viable dominant microorganisms responsible for the traditional brewing of Hong Qu glutinous rice wine

Cited by 39 publications

References 38 publications

Strand Displacement Amplification for Multiplex Detection of Nucleic Acids

Strand Displacement Amplification for Multiplex Detection of Nucleic Acids

Detection of Viable and Total Bacterial Community in the Pit Mud of Chinese Strong-Flavor Liquor Using Propidium Monoazide Combined With Quantitative PCR and 16S rRNA Gene Sequencing

Real-Time PCR and Quantitative Culture for Mycoplasma pneumoniae Load in Pharyngeal Swabs from Children at Preliminary Diagnosis and Discharge

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