A comparison of nucleic acid amplification techniques for the assessment of bacterial viability

Birch, L.; Dawson, Carolyn; Cornett, Johanne H.; Keer, Jacquie T.

doi:10.1046/j.1472-765x.2001.00999.x

Cited by 102 publications

(50 citation statements)

References 22 publications

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“…13 In addition, some authors noted that mRNA can persist for an extended period after cell death, precluding their use to detect bacterial viability. [14][15][16] Like PMA, ethidium bromide monoazide (EMA) is known as a DNA intercalating agent, and its use has been reported for discriminating intact from dead bacteria by PCR. [17][18][19] However, recent reports have suggested that EMA can influence intact cells as well as dead cells in some bacterial strains, and that EMA is not a suitable indicator of bacterial viability.…”

Section: Discussionmentioning

confidence: 99%

Improving clinical significance of PCR: Use of propidium monoazide to distinguish viable from dead staphylococcus aureus and staphylococcus epidermidis

Kobayashi

Oethinger

Tuohy

et al. 2009

Journal Orthopaedic Research

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Molecular techniques, such as the polymerase chain reaction (PCR) have high sensitivity when used to diagnose infection, but may detect DNA, RNA, and proteins from dead, as well as viable, bacteria. Propidium monoazide (PMA) is a DNA binding agent, that has the ability to penetrate only dead cells with compromised membranes and has been used in conjunction with real-time PCR to distinguish intact from dead bacterial cells. In this study, intact, heat-inactivated (dead), and intact/dead admixed Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis) were treated with PMA or left untreated before DNA extraction. We quantified levels of 16S rDNA and tuf gene by real-time quantitative PCR (qPCR), to test the ability of PMA to distinguish intact from dead bacteria. Our results indicated that PMA inhibited detection of dead bacteria, and the qPCR results reflected the number of intact bacteria without being impacted by the presence of the dead bacteria. This approach of combining qPCR with and without PMA treatment has promise to limit false-positive PCR results when used to diagnose infections, but needs to be further validated in clinical samples. ß

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Section: Discussionmentioning

confidence: 99%

Improving clinical significance of PCR: Use of propidium monoazide to distinguish viable from dead staphylococcus aureus and staphylococcus epidermidis

Kobayashi

Oethinger

Tuohy

et al. 2009

Journal Orthopaedic Research

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“…However, it is important to note that the detection of M. leprae DNA does not indicate that the bacilli in the analyzed environmental water samples are viable. The detected DNA may originate from live or dead bacteria, or even constitute extracellular DNA 23 ; detection of bacterial DNA derived from lysis by heating followed by ultraviolet exposure has been demonstrated 24,25 .…”

Section: Tablementioning

confidence: 99%

Presence of Mycobacterium leprae genotype 4 in environmental waters in Northeast Brazil

Holanda

Marques

Macedo

et al. 2017

Rev. Soc. Bras. Med. Trop.

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Introduction: This study quantified Mycobacterium leprae bacilli in environmental water samples from five municipalities in the State of Ceará by quantitative polymerase chain reaction (qPCR) and compared the identified genotypes with those obtained from leprosy patient biopsies. Methods: We collected five replicas from each of the 30 selected reservoirs and skin lesion biopsies from 25 new leprosy cases treated at a reference center in Fortaleza, Ceará from 2010 to 2013. The 16S rRNA gene region of M. leprae was amplified by qPCR and a standard curve was created with the pIDTBlue 16SrRNAMlep plasmid. The Juazeiro do Norte water samples and the biopsies were genotyped (single nucleotide polymorphism [SNP] 1 to 4) and the SNP 4 genotypes were subtyped. Results: Of the 149 water samples analyzed, 54.4% were positive for the M. leprae DNA. The M. leprae bacilli copy number ranged from 1.42 × 10 -1 to 1.44 × 10 + 2 . Most biopsies showed SNP type 4 (64%), while all samples from Juazeiro do Norte were SNP type 4, with subtype 4-N appearing at the highest frequency. Conclusions: We suggest that environmental waters containing M. leprae bacilli play an important role in disease transmission, justifying PGL-1 seropositivity in individuals living in areas where there is no reported case, and in leprosy cases individuals who report no previous contact with other case. Therefore, further investigation is needed to clarify disease transmission in this region and to explore the role of the environment. We also suggest that in this area surveillance for leprosy cases should be intensified.

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“…The disadvantages of the assay include: i) the presence of possible contamination with genomic DNA, which often leads to false-positive results; and ii) some RNA molecules may persist in cells in a detectable form for an extended time period (about 1 h) after loss of cell viability in some specific conditions, which may lead to false positive results [101].…”

Section: Reverse Transcription Polymerase Chain Reaction (Rt-pcr)mentioning

confidence: 99%

Recent and the Latest Developments in Rapid and Efficient Detection of Salmonella in Food and Water

Hu¹,

Li²

2017

Adv Tech Biol Med

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Salmonella is a leading cause of foodborne diseases. Gastroenteritis caused by nontyphoid Salmonella is still a major infectious disease in the world. About 95% of Salmonella infections are caused by ingestion of contaminated food and water. Rapid, sensitive, and efficient detection and identification of Salmonella from foods and water are critical for minimizing the spread of outbreaks caused by this pathogen. These methods can be applied to track the food source of contamination or by early diagnosis of the infections in a clinical setting. Culture-based methods for detection of Salmonella are laborious and time-consuming, typically taking 5-7 days to obtain a pure culture and serovar identification. In the past few decades, molecular-based technologies have greatly shortened the time for detection and identification of bacterial pathogens from food and water, and drastically increased the specificity and sensitivity of the assays. In this review, we report an update on the development of rapid and efficient methods for the detection and identification of Salmonella in food and water, focusing on the approaches for concentration of Salmonella cells and viable cell detection, as well as the advantages and drawbacks of these methods.

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A comparison of nucleic acid amplification techniques for the assessment of bacterial viability

Cited by 102 publications

References 22 publications

Improving clinical significance of PCR: Use of propidium monoazide to distinguish viable from dead staphylococcus aureus and staphylococcus epidermidis

Improving clinical significance of PCR: Use of propidium monoazide to distinguish viable from dead staphylococcus aureus and staphylococcus epidermidis

Presence of Mycobacterium leprae genotype 4 in environmental waters in Northeast Brazil

Recent and the Latest Developments in Rapid and Efficient Detection of Salmonella in Food and Water

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