Taq DNA polymerase is the most widely used enzyme for PCR amplification and it contains 832 amino acids but no cysteines. Bovine serum albumin (BSA) is an 832-amino acid protein containing 35 cysteines, and at high temperature BSA can produce protein hydrogels through recombined disulfides and noncovalent bonds. When hydrogels are prepared using a mixture of Taq DNA polymerase and BSA, no disulfide bonds occur between them, and noncovalent bonds are the main forces holding together the Taq DNA polymerase with the hydrogels. During PCR amplification, temperature cycling can destroy the noncovalent bonds, thereby releasing Taq DNA polymerase from the hydrogels to synthesize DNA. The release rate, regulated through the variation of BSA concentration, allows for velocity-controlled amplification of DNA. As the amount of DNA products exceeds that of the Taq DNA polymerase released from hydrogels, a linear amplification replaces the exponential PCR amplification. When the amplification velocity begins to decrease, the time needed for reaching a plateau increase. This provides a broader detection window for the analysis of the end products, which correspond to the difference compared with the initial DNA concentration. The number of primers is continuously reduced with increasing cycles, which further causes a decline in the DNA amplification efficiency. The difference in the initial DNA template may cause a different consumption of primers, which can cause errors in the Ct values in the case of qPCR. Based on the slopes of the linear amplification curve, the Ct values can be calibrated to reduce this error, and a more accurate detection of the initial content of the target DNA can be implemented.
Ning Ding,Ziwei Li,Lin Jiang,Hong Liu,Yanping Zhang,Yingxue Sun
Performic acid (PFA) has been increasingly used in wastewater disinfection due to its strong oxidizing ability and few disinfection byproducts. However, its disinfection pathways and mechanisms towards pathogenic bacteria disinfection are poorly understood. In this study, E. coli , S. aureus , and B. subtilis were inactivated using sodium hypochlorite (NaClO), PFA, and peracetic acid (PAA) in simulated turbid water and municipal secondary effluent. Cell culture-based plate counting showed that E. coli and S. aureus were extremely susceptible to NaClO and PFA and achieved a 4-log inactivation at CT s ≤ 1 mg/L·min with an initial disinfectant concentration of 0.3 mg/L. B. subtilis was much more resistant. At the initial disinfectant dose of 7.5 mg/L, PFA required CT s of 3–13 mg/L·min to achieve a 4-log inactivation. Turbidity negatively affected the disinfection. In the secondary effluent, the CT s required for PFA to achieve a 4-log inactivation of E. coli and B. subtilis were 6–12 times higher than those required in simulated turbid water, and a 4-log inactivation of S. aureus could not be achieved. PAA showed a much weaker disinfection ability than the other two disinfectants. The reaction pathways of E. coli inactivation by PFA included both direct and indirect reactions, in which the PFA molecule accounted for 73 %, and ·OH and peroxide radicals accounted for 20 % and 6 %, respectively. During PFA disinfection, E. coli cells were severely disintegrated, while the S. aureus cell exteriors remained mostly intact. B. subtilis was the least affected. Compared with cell culture-based analysis, the inactivation detected by flow cytometry was significantly lower. Viable but non-culturable bacteria after disinfection were believed to be primarily responsible for this inconsistency. This study suggested that PFA was able to control regular bacteria in wastewater, but it should be used with caution when treating recalcitrant pathogens.
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