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.

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 MOLECULAR DETECTION OF ASTROVIRUS USING REVERSE TRANSCRIPTASE QUANTITATIVE POLYMERASE CHAIN REACTION TECHNIQUE

2020 ◽  
Vol 51 (Special) ◽  
Author(s):  
Fadhil & et al.
Keyword(s):  
Reverse Transcriptase ◽  
Molecular Detection ◽  
Quantitative Polymerase Chain Reaction ◽  
Melting Curve ◽  
Single Infection ◽  
Chain Reaction ◽  
Qpcr Analysis ◽  
Efficient Detection ◽  
Stool Samples ◽  
Polymerase Chain

This study was aimed to highlight the importance of the melt curve-quantitative reverse transcriptase PCR (RT-qPCR) analysis in the detection of astrovirus (AstV) from both negative and positive rotavirus and enterovirus (EVs) samples, and the effectiveness of the AstV infection on the vaccine immunogenicity in the vaccinated infected children.  By RT-qPCR based Sybre green associated- melting curve assay, stool samples of 49 enterovirus suspected patients and of 39 rotavirus suspected patients were tested for AstV. Results of EVs group showed a 29 (59.2%) positive AstV contributed to 26 (89.5%) co-infection with Evs and 3 (10.3%) as a single infection in negative samples for Evs. Furthermore, AstV co-infection percentage is higher than the single infection. Moreover, the percentage of the Astrovirus among the vaccinated AFP-suspected cases was 53%, while the percentage of these viruses among the unvaccinated was 100%. Thus, MamAstrovirus- 1 MK948878 is the first local isolate recorded in the Genbank. In conclusion, the RT-qPCR based on SYBR Green showed the rapid and efficient detection of AstV with few copies number. This allow to be used for the diagnosis of AstV along with other gastroenteritis viruses in a multiplex assay to reduce processing time.

scholarly journals Anti-tumour effect of tocilizumab for osteosarcoma cell lines

2020 ◽  
Vol 9 (11) ◽  
pp. 821-826
Author(s):  
Tomohito Hagi ◽  
Tomoki Nakamura ◽  
Kouji Kita ◽  
Takahiro Iino ◽  
Kunihiro Asanuma ◽  
...  
Keyword(s):  
Cell Lines ◽  
Quantitative Polymerase Chain Reaction ◽  
Osteosarcoma Cell ◽  
Preclinical Models ◽  
Conventional Chemotherapy ◽  
Chain Reaction ◽  
Qpcr Analysis ◽  
Polymerase Chain ◽  
Proliferation And Invasion ◽  
Anti Tumour Effect

Aims Tocilizumab, an interleukin-6 (IL-6) receptor (IL-6R) targeting antibody, enhances the anti-tumour effect of conventional chemotherapy in preclinical models of cancer. We investigated the anti-tumour effect of tocilizumab in osteosarcoma (OS) cell lines. Methods We used the 143B, HOS, and Saos-2 human OS cell lines. We first analyzed the IL-6 gene expression and IL-6Rα protein expression in OS cells using reverse transcription real time quantitative-polymerase chain reaction (RT-qPCR) analysis and western blotting, respectively. We also assessed the effect of tocilizumab on OS cells using proliferation and invasion assay. Results The OS cell lines 143B, HOS, and Saos-2 expressed IL-6R. Recombinant human IL-6 treatment increased proliferation of 143B and HOS cells. Tocilizumab treatment decreased proliferation and invasion of 143B, HOS, and Saos-2. Conclusion In conclusion, we confirmed the production of IL-6 and the expression of IL-6R in OS cells and demonstrated that tocilizumab inhibits proliferation and invasion in OS cells. Cite this article: Bone Joint Res 2020;9(11):821–826.

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