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Genes and genomics
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2018
Nested PCR is normally used to increase the specificity of DNA amplification, by reducing background due to nonspecific amplification of DNA. Two sets of primers are used in two successive PCRs. In the first reaction, one pair of primers is used to generate DNA products, which, besides the intended target, may still consist of nonspecifically amplified DNA fragments. The product(s) are then used in a second PCR with a set of primers whose binding sites are completely or partially different from and located at 3′ of each of the primers used in the first reaction. Nested PCR is often more successful in specifically amplifying long DNA fragments than conventional PCR, but it requires more detailed knowledge of the target sequences.
Diagnostics
Published in Ronald Fayer, Lihua Xiao, Cryptosporidium and Cryptosporidiosis, 2007
In these instances, and particularly when clinical suspicion is high, oocyst-negative stool samples should be subjected to antigen and PCR-based detection, because sufficient Cryptosporidium antigen or DNA from asexual life-cycle forms can be present in feces. For PCR-based methods, nested PCR methods, being more sensitive than direct PCR methods, are likely to have a higher diagnostic index.
Dual-target one-step nested PCR for sensitive detection of SARS-CoV-2 nucleic acids
Published in Preparative Biochemistry & Biotechnology, 2022
Qijie Li, Yiqing Xia, Dunshui Liao, Hu Nie, Ming Zhang, Tinghua Wang, Jiayu Liao, Qingjie Xia
The PCR products synthesized by the outer-outer, outer-inner, and inner-outer primers in the DTO-N-PCR system are all additional templates for the inner-inner primers. The PCR amplification speed guided only by the inner primers was more than twice, the theoretical value of the PCR products after n cycles would thus be as the amount of the initial template, i.e., 1/8 (n2+3n) 2n times (Figure 3(B)). Notably, the sum of PCR products guided by all primers would be 1/8 (n2+7n + 4) 2n times the initial template amount (Figure 3(B)). In contrast, the theoretical value after n cycles in the conventional RT-qPCR was 2n times the initial template amount. The amplification efficiency E of the DTO-N-PCR was 137.9%, indicating that the amplification rate was greater than two times. Moreover, some fluorescence could still be detected when the mixed DNA template was diluted to 0.0002 pg to correspond to about 26 copies of each positive control DNA, indicating the high sensitivity of the DTO-N-PCR system. Nonetheless, the Ct values of DTO-N-PCR were lower than those of conventional RT-qPCR in detecting SARS-CoV-2 positive clinical pharyngeal specimens. These results indicate that the DTO-N-PCR system can greatly improve the amplification efficiency and enhance the sensitivity of SARS-CoV-2 detection. Moreover, the design of the inner and outer primers of the nested PCR and specific probes enhances its accuracy and specificity.