scholarly journals Viability determination of Ascaris ova in raw wastewater: a comparative evaluation of culture-based, BacLight Live/Dead staining and PMA-qPCR methods

2019 ◽  
Vol 80 (5) ◽  
pp. 817-826 ◽  
Author(s):  
Vivek B. Ravindran ◽  
Esmaeil Shahsavari ◽  
Sarvesh K. Soni ◽  
Andrew S. Ball
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Comparative Assessment ◽  
Detection Methods ◽  
Propidium Monoazide ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Potential Public Health ◽  
Viability Determination ◽  
Raw Wastewater

Abstract Accurate evaluation of viable Ascaris ova in wastewater is the key to mitigating Ascaris reinfections in endemic regions. In this study, the viability of Ascaris ova in raw wastewater was determined using three different detection methods: culture-based, BacLight Live/Dead staining and propidium monoazide–quantitative polymerase chain reaction (PMA-qPCR). Furthermore, comparative assessment of viability utilising the aforementioned detection methods was performed using seeded experiments in wastewater. The percentage of viability was: culture-based (82%), BacLight Live/Dead staining (87%) and PMA-qPCR (85%) respectively. Despite the fact that no statistical difference was shown in the viability determination among the three methods, PMA-qPCR-based viability determination would be preferable over the other two methods for evaluating potential public health risks with A. suum ova due to its accuracy, being least subjective and its rapid reaction time.

scholarly journals Comparison of culture-based, vital stain and PMA-qPCR methods for the quantitative detection of viable hookworm ova

2017 ◽  
Vol 75 (11) ◽  
pp. 2615-2621 ◽  
Author(s):  
P. Gyawali ◽  
J. P. S. Sidhu ◽  
W. Ahmed ◽  
P. Jagals ◽  
S. Toze
Keyword(s):  
Environmental Samples ◽  
Quantitative Measurement ◽  
Quantitative Polymerase Chain Reaction ◽  
Detection Methods ◽  
Quantitative Detection ◽  
Propidium Monoazide ◽  
Chain Reaction ◽  
Vital Stain ◽  
Polymerase Chain ◽  
Hookworm Ova

Accurate quantitative measurement of viable hookworm ova from environmental samples is the key to controlling hookworm re-infections in the endemic regions. In this study, the accuracy of three quantitative detection methods [culture-based, vital stain and propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR)] was evaluated by enumerating 1,000 ± 50 Ancylostoma caninum ova in the laboratory. The culture-based method was able to quantify an average of 397 ± 59 viable hookworm ova. Similarly, vital stain and PMA-qPCR methods quantified 644 ± 87 and 587 ± 91 viable ova, respectively. The numbers of viable ova estimated by the culture-based method were significantly (P < 0.05) lower than vital stain and PMA-qPCR methods. Therefore, both PMA-qPCR and vital stain methods appear to be suitable for the quantitative detection of viable hookworm ova. However, PMA-qPCR would be preferable over the vital stain method in scenarios where ova speciation is needed.

scholarly journals Technical Validation of a Reverse-Transcription Quantitative Polymerase Chain Reaction In Vitro Diagnostic Test for the Determination of MiR-31-3p Expression Levels in Formalin-Fixed Paraffin-Embedded Metastatic Colorectal Cancer Tumor Specimens

2018 ◽  
Vol 13 ◽  
pp. 117727191876335 ◽  
Author(s):  
Lucas Ramon ◽  
Catherine David ◽  
Karine Fontaine ◽  
Elodie Lallet ◽  
Charles Marcaillou ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Quantitative Polymerase Chain Reaction ◽  
Chain Reaction ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Polymerase Chain ◽  
Formalin Fixed

MiR-31-3p expression has been shown to be a predictive biomarker for response to anti-epithelial growth factor receptor therapy in patients with RAS wild-type metastatic colorectal cancer (mCRC). To aid in the quantification of miR-31-3p expression in formalin-fixed paraffin-embedded (FFPE) primary tumor samples from patients with mCRC, a reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay was developed and validated. Assay development included the identification of a microRNA reference standard and the determination of an appropriate relative quantification cutoff for differentiating low versus high miR-31-3p expression. Sample specimens for the validation studies included both FFPE slides and shavings. Polymerase chain reaction (PCR) efficiency and linearity, analytical sensitivity and specificity, assay robustness, reproducibility, and accuracy were demonstrated across a number of test conditions and differing quantitative PCR platforms. The data from this study provide evidence as to the feasibility of quantifying the expression of miR-31-3p from FFPE tumor tissue using a standardized RT-qPCR assay.

scholarly journals Propidium monoazide combined with qPCR to differentiate live and dead conidia of Neofabraea actinidiae

2018 ◽  
Vol 71 ◽  
pp. 348
Author(s):  
Lucia Ramos ◽  
I.P. Shamini Pushparajah ◽  
M. Shahjahan Kabir ◽  
Bethan E. Parry ◽  
Kerry R. Everett
Keyword(s):  
Light Emitting Diode ◽  
Quantitative Polymerase Chain Reaction ◽  
Propidium Monoazide ◽  
Chain Reaction ◽  
Light Emitting ◽  
Qpcr Analysis ◽  
Low Concentrations ◽  
Polymerase Chain ◽  
High Concentrations ◽  
Commercial Kit

Neofabraea actinidiae can occasionally cause post-harvest rot in kiwifruit. Quantitative polymerase chain reaction (qPCR) analysis represents a feasible and accurate option for identifying and quantifying this rot but is limited because qPCR results do not differentiate live and dead conidia. Propidium monoazide (PMA) is a photoreactive dye that penetrates into the damaged cell-wall membranes of dead conidia binding to the DNA and thus suppressing its amplification by qPCR. A commercial kit containing PMA was trialled for differentiating between live and dead N. actinidiae conidia. The most suitable conditions were 1 μM PMA with 10 min light emitting diode (LED) exposure, and could clearly distinguish high concentrations of live from similar concentrations of dead conidia when tested separately and as a mixture. Low concentrations of live N. actinidiae conidia could be distinguished from dead ones when tested separately, but not as a mixture. Additional work is needed to optimise the effectiveness of the PMA binding and apply this concept in the orchard.

Rapid and accurate determination of the potency of varicella vaccine by quantitative polymerase chain reaction

Vaccine ◽  
2011 ◽  
Vol 29 (47) ◽  
pp. 8490-8495 ◽  
Author(s):  
Marsha S. Russell ◽  
Changgui Li ◽  
Louise Larocque ◽  
Junzhi Wang ◽  
Aaron Farnsworth ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Quantitative Polymerase Chain Reaction ◽  
Accurate Determination ◽  
Varicella Vaccine ◽  
Chain Reaction ◽  
Polymerase Chain
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