Optimization of multiplex quantitative polymerase chain reaction based on response surface methodology and an artificial neural network-genetic algorithm approach

Pan, Peng; Jin, Weifeng; Li, Xiaohong; Chen, Yi; Jiang, Jiahui; Wan, Haitong; Yu, Dan

doi:10.1371/journal.pone.0200962

Cited by 9 publications

(5 citation statements)

References 36 publications

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“…Hence, we developed a TaqMan-based fast multiplex quantitative real-time PCR assay for the simultaneous detection of dog cox1, human cytb and Leishmania kDNA in female sand flies. The addition of multiple primers and probes in a single reaction as well as changes in the number of cycles and annealing temperature can affect the specificity, sensitivity and efficiency of real-time PCR assays [23,24]. This is in fact one of the main obstacles to overcome while developing a multiplex real-time PCR assay [17].…”

Section: Discussionmentioning

confidence: 99%

Fast multiplex real-time PCR assay for simultaneous detection of dog and human blood and Leishmania parasites in sand flies

et al. 2020

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Background: The blood-feeding behaviour of female sand flies may increase their likelihood of acquiring and transmitting Leishmania parasites. Studies on the host usage by these insects may thus improve our understanding of the Leishmania transmission risk in leishmaniasis-endemic areas. Here, we developed a fast multiplex real-time PCR assay for simultaneous detection of dog, human and Leishmania DNA in sand flies. Methods: Primers and TaqMan probes targeting the mitochondrial cytochrome c oxidase subunit 1 and cytochrome b genes of dog and human, respectively, were combined in a multiplex assay, which also includes primers and a TaqMan probe targeting the Leishmania minicircle kinetoplast DNA. Results: The multiplex assay was 100% specific, with analytical sensitivities of 10 3 fg/reaction for dog and human and 1 fg for Leishmania. By testing field-collected engorged female sand flies (95 Migonemyia migonei and two Nyssomyia intermedia), 50 M. migonei were positive for one or two targets (positivity rates: 45.4% for human, 4.1% for dog and 12.4% for Leishmania DNA). Conclusions: This multiplex real-time PCR assay represents a novel fast assay for detecting dog, human and Leishmania DNA in female sand flies and therefore a tool for assessing the risk of Leishmania transmission to these hosts in areas of active transmission.

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Section: Discussionmentioning

confidence: 99%

Fast multiplex real-time PCR assay for simultaneous detection of dog and human blood and Leishmania parasites in sand flies

et al. 2020

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“…RSM, an effective statistical method and mathematical techniques in optimizing complex systems, is usually used to obtain the optimal parameters by constructing mathematical models and analysis of regression and variance [15,16,17]. By using RSM, the number of experimental trials was reduced and the interactions between independent variables were illustrated [18,19].…”

Section: Resultsmentioning

confidence: 99%

Optimization of Degradation Conditions with PRG, a Polysaccharide from Phellinus ribis, by RSM and the Neuroprotective Activity in PC12 Cells Damaged by Aβ25–35

et al. 2019

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In the previous work, we found PRG, a polysaccharide from Phellinus ribis, exhibited neurotrophic activity. To obtain an active structural unit with lower molecular weight, PRG was degraded to prepare the degraded PRG (DPRG) using ascorbic acid and H2O2. The aim of the paper was to obtain DPRG by optimizing the degradation conditions using response surface methodology (RSM) and to study its protective effects of PC12 cells induced by Aβ25–35. The optimum conditions were as follows; the concentration of H2O2-Vc was 17 mM and degradation temperature was 50 °C; when degradation time was 1.6 h, the experimental response value of PC12 cell viability was 83.4 ± 0.15%, which was in accordance with the predicted value (83.5%). We also studied the protective effects of DPRG against the Aβ25–35-induced neurotoxicity and explored the underlying mechanism. The results showed that treatment with DPRG could attenuate PC12 cells death. The mechanism was relative to the inhibition of cell apoptosis by increasing the MMP level and decreasing the protein expression of cytochrome C (Cytc) in PC12 cells. In conclusion, DPRG with lower molecular weight was obtained successfully. It possessed neuroprotective properties and might be a candidate for neurodegenerative disease treatment.

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“…2013; Pan et al . 2018). We used single primer pairs to reduce the complexity and optimised the ratio of primers and probes for each species.…”

Section: Discussionmentioning

confidence: 99%

Development and use of a single real‐time PCR assay to identify the three spider mite species Tetranychus urticae, Tetranychus lambi and Tetranychus ludeni (Acari: Tetranychidae)

et al. 2020

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Tetranychid spider mites are a significant worldwide pest of agriculture with Tetranychus urticae being the most important. In Australian cotton, T. urticae is the most damaging, but Tetranychus ludeni and Tetranychus lambi are also present and can be difficult to distinguish morphologically. For this reason, a fast‐diagnostic assay was developed to identify T. urticae, T. lambi and T. ludeni in a single real‐time PCR reaction. The assay design was based on a universal primer pair to amplify the internal transcribed spacer of nuclear ribosomal DNA (ITS1) with three species‐specific TaqMan probes. The assay was validated by sequencing three known reference strains for T. urticae, T. lambi and T. ludeni and a blind test on 14 DNA samples that included species other than the three target species. The method correctly identified the target species and produced a negative result against other non‐target species. Furthermore, we examined 22 field‐collected spider mite populations from Australian cotton that had been laboratory maintained. Here, we found that 50% of the populations labelled as T. urticae were mixed with T. lambi and quarter of the populations labelled with T. lambi contained T. urticae. It demonstrates just how difficult it is to maintain strain integrity with multiple species in proximity. Finally, the assay was used by Biosecurity Australia to test 2125 mite quarantine intercept specimens. They found 1949 T. urticae, 1 T. lambi, 13 T. ludeni and 162 unknown specimens confirming assay accuracy and robustness. The diagnostic assay developed can provide quick spider mite species identification and can support multiple uses including species identification to support spray decisions or the precise identification of border quarantine intercepts where accuracy and turnaround time is critical.

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Optimization of multiplex quantitative polymerase chain reaction based on response surface methodology and an artificial neural network-genetic algorithm approach

Cited by 9 publications

References 36 publications

Fast multiplex real-time PCR assay for simultaneous detection of dog and human blood and Leishmania parasites in sand flies

Fast multiplex real-time PCR assay for simultaneous detection of dog and human blood and Leishmania parasites in sand flies

Optimization of Degradation Conditions with PRG, a Polysaccharide from Phellinus ribis, by RSM and the Neuroprotective Activity in PC12 Cells Damaged by Aβ25–35

Development and use of a single real‐time PCR assay to identify the three spider mite species Tetranychus urticae, Tetranychus lambi and Tetranychus ludeni (Acari: Tetranychidae)

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