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Isothermal Amplification of RNA and Its Application in Clinical Samples
Author: LiuBing
Tutor: ChenChao
School: Northwestern University
Course: Microbiology
Keywords: HBV S gene mRNA HBV DNA Magnetic particles Isothermal amplification
CLC: R450
Type: Master's thesis
Year: 2007
Downloads: 104
Quote: 0
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Abstract
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Phenomenon of input containing infected blood or blood products infected with HIV , hepatitis B , hepatitis C and other diseases , has caused a high degree of social attention . The most important task is to strictly control the source of foreign blood screening , traditional ELISA method to detect blood-borne diseases are still different limitations , such as low detection flux , time consuming , and low sensitivity . Quantitative PCR assay detection of the high cost of the equipment requirements of the disadvantages is not conducive to the promotion of grassroots blood banks and hospitals . In this study, isothermal amplification technology, biotin - streptavidin and biotin system enrichment of target sequences , and aims to establish a high sensitivity and specificity of detection of low copy number nucleic acid samples . . This study yeast total RNA was extracted from the HBV S gene expression vector the GS115-pPICZS yeast cells . Capture probes coupled streptavidin magnetic particles , by biotin avidin - chain reaction , and is connected to the target nucleotide , the enrichment of a sample of HBV S gene mRNA . Isothermal amplification amplification HBVS gene mRNA . The amplification product was detected by electrophoresis on a 1.5% agarose gel . Optimize isothermal amplification system , the detection of HBV S gene mRNA amplification factor up to the order of magnitude of 10 to 8 , sensitivity can reach 2pg/mL , higher than the reverse transcriptase PCR 200 pg / mL sensitivity . The application of the Institute in the development of magnetic particles enriched target sequence and isothermal amplification technology , selected by the fluorescent quantitative PCR analysis of HBV DNA levels greater than 1.0 × 10 ~ 3 copies / mL serum samples for the detection of HBV DNA results for HBV detection of HBV molecular biology, molecular epidemiology study has important significance . Isothermal amplification reaction system with good sensitivity and specificity in the detection of HBV DNA in HBV S gene mRNA and serum specimens . Not only can effectively detect HBV S gene mRNA and serum samples HBV DNA also has an important value and significance , and for the detection of low copy number nucleic acid samples . The technology has the potential to replace PCR technology has become the primary means of amplification of nucleic acid samples .
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