High rates of new cervical cancer cases and deaths occur in low- and middle-income countries yearly, and one reason was found related to limitation of regular cervical cancer screening in local and low-resource settings. HPV has over 150 types, yet certain 14–20 high-risk and 13–14 low-risk types are common, and, thus, most conventional HPV nucleic acid assays, for examples, Cobas 4800 HPV test (Roche Diagnostics, New Jersey, USA) and REBA HPV-ID (Molecules and Diagnostics, Wonju, Republic of Korea) were developed to cover these types. We thereby utilized bioinformatics combined with recent isothermal amplification technique at 35–42 °C to firstly describe multiplex recombinase polymerase amplification assay that is specific to these common 20 high-risk and 14 low-risk types, and also L1 and E6/E7 genes that target different stages of cervical cancer development. Multiplex primer concentrations and reaction incubation conditions were optimized to allow simultaneous two gene detections at limit of detection of 1000 copies (equivalent to 2.01 fg) for L1 and 100 copies (0.0125 fg) for E6/E7, respectively. The assay was validated against urogenital and other pathogens, normal flora, and human control. In 130 real clinical sample tests, the assay demonstrated 100% specificity, 78% diagnostic accuracy, and 75% sensitivity compared with REBA HPV-ID test, and is much more rapid (15–40 min), less expensive (~ 3–4 USD/reaction) and does not require instrumentation (35–42 °C reaction condition so hand holding or tropical temperature is possible). Hence, the developed novel assay provides alternative screening tool for potential local screening. Furthermore, as this assay uses safe chemical reagents, it is safe for users.
Cassava is one of the most important starch crops in the world. Cassava starch factories normally generate a huge amount of cassava tuberous root residual which is usually discarded and might cause pollution to the environment. In order to find some extra benefits of such waste, in this study, cassava root cortex peroxidase (CCP) was found up to 20 mg/kg fresh deteriorated cortex tissue from tuberous root and also able to demonstrate some applications similar to horseradish peroxidase (HRP). The characterization revealed that major native CCP was a 105-kDa dimeric peroxidase with two 54-kDa monomers. Using 3,3′-diaminobenzidine (DAB) as substrate in the assay, CCP was found to be tolerant and could maintain its activity in a wide temperature range from 20 to 70°C with an optimum at 65°C. CCP was stable in board pH range from 3 to 11 with maximum activity at pH 5.0. Despite simple purification with ammonium sulfate precipitation, partial purified CCP was capable of determining glucose concentrations with glucose oxidase as similar capability as horseradish peroxidase (HRP). For application as reporter enzyme in immunoassays, the self-made secondary antibody conjugated with CCP did successfully detect the protein antigen in Western blot analysis using the luminol as chemiluminescent substrate. These demonstrations indicated CCP as one of the most robust peroxidases. Moreover, the active enzyme could be easily retrieved from the industrial waste of cassava peel at low cost. Further studies should involve optimization of enzyme purification in industrial scale and finding more CCP potential applications which should increase the advantages of this promising enzyme.
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