HSV-1, HSV-2. CMV, EBV의 빠르고 정확한 검출을 위한 multiplex PCR 방법의 개발은 임상적으로 환자의 치료를 위해 매우 중요하다. 바이러스의 multiplex PCR 검출법은 적은 비용과 시간으로 많은 시료를 진단할 수 있다. multiplex PCR을 forward와 reverse primers의 annealing 온도, primers의 ...
HSV-1, HSV-2. CMV, EBV의 빠르고 정확한 검출을 위한 multiplex PCR 방법의 개발은 임상적으로 환자의 치료를 위해 매우 중요하다. 바이러스의 multiplex PCR 검출법은 적은 비용과 시간으로 많은 시료를 진단할 수 있다. multiplex PCR을 forward와 reverse primers의 annealing 온도, primers의 self-annealing 및 증폭산물들의 크기가 서로 겹치지 않도록 조건을 갖추어야 한다. 따라서 quadruplex PCR은 1.5mM MgCl_(2), 200uM dNTPs와 HSV-1(2pmoles), HSV-2(1.5pmoles), CMV(1pmoles), EBV(1pmoles) 농도의 저건에서 반응이 잘 일어났으며 이때 primers 들의 annealing 온도는 54℃이었다. 이 조건에서 10 copies의 재조합 plasmid DNA에서도 검출이 가능하였는데 재조합 plasmid DNA는 vector에 target DNA의 PCR 산물을 cloning하여 사용하였다. PCR 증폭산물들의 크기는 HSV-1(271bp), HSV-2(231bp), CMV(368bp), EBV(326bp)의 크기가 생성되도록 고안하였고, 2% Agarose gel에서 서로 분리가 되었으며 염기서열 분식을 통하여 확인하였다. 여기서 확립된 quadruplex PCR법은 HSV-1, HSV-2, CMV, EBV의 빠르고 정확한 검출을 필요로 할 때 임상적 진단에 매우 유용하게 사용될 수 있음을 시사한다.
HSV-1, HSV-2. CMV, EBV의 빠르고 정확한 검출을 위한 multiplex PCR 방법의 개발은 임상적으로 환자의 치료를 위해 매우 중요하다. 바이러스의 multiplex PCR 검출법은 적은 비용과 시간으로 많은 시료를 진단할 수 있다. multiplex PCR을 forward와 reverse primers의 annealing 온도, primers의 self-annealing 및 증폭산물들의 크기가 서로 겹치지 않도록 조건을 갖추어야 한다. 따라서 quadruplex PCR은 1.5mM MgCl_(2), 200uM dNTPs와 HSV-1(2pmoles), HSV-2(1.5pmoles), CMV(1pmoles), EBV(1pmoles) 농도의 저건에서 반응이 잘 일어났으며 이때 primers 들의 annealing 온도는 54℃이었다. 이 조건에서 10 copies의 재조합 plasmid DNA에서도 검출이 가능하였는데 재조합 plasmid DNA는 vector에 target DNA의 PCR 산물을 cloning하여 사용하였다. PCR 증폭산물들의 크기는 HSV-1(271bp), HSV-2(231bp), CMV(368bp), EBV(326bp)의 크기가 생성되도록 고안하였고, 2% Agarose gel에서 서로 분리가 되었으며 염기서열 분식을 통하여 확인하였다. 여기서 확립된 quadruplex PCR법은 HSV-1, HSV-2, CMV, EBV의 빠르고 정확한 검출을 필요로 할 때 임상적 진단에 매우 유용하게 사용될 수 있음을 시사한다.
The development of a multiplex polymerase chain reaction method for the rapid and accurate detedtion and typing of HSV-1. HSV-2, CMV and EBV is very important for clinical diagnosis to deliver therapy as early as possible. A large scale amplifications by multiplex PCR of viral DNA could lower the co...
The development of a multiplex polymerase chain reaction method for the rapid and accurate detedtion and typing of HSV-1. HSV-2, CMV and EBV is very important for clinical diagnosis to deliver therapy as early as possible. A large scale amplifications by multiplex PCR of viral DNA could lower the cost and time for viral diagnosis. However, one has to consider various problems concerning PCR conditions such as annealing temperature of the forward and reverse primers of each virus, and avoidance of the self-annealing of primers and the size overlapping of PCR products for exact base calling. Thus, sensitive quadruplex PCR can be achieved by optimizing parameters such as primers, 1.5mM magnesium and 200 uM dNTPs concentrations. The concentration of HSV-1, HSV-2, CMV and EBV primers were 2.0 pmoles, 1.5 pmoles, 1.0 pmoles and 1.0 pmoles, respectively. Optimal annealing temperature was 54℃. And by this method we could detect 10 copies of reconstructed template plasmid DNA which were cloned to vectors containing target sequences of viral DNA, PCR products of HSV-1 having 271 bp, 231 bp for HSV-2, 368 bp for CMV, and 326 bp for EBV were separated on 2.0% agarose gel electrophoresis and confirmed by direct sequencing, The present study showed that quadruplex PCR assay described herein has a potentiality for application in clinical diagnosis, when a rapid and accurate detection, and typing of viruses HSV-1, HSV-2, CMV or EBV are necessary.
The development of a multiplex polymerase chain reaction method for the rapid and accurate detedtion and typing of HSV-1. HSV-2, CMV and EBV is very important for clinical diagnosis to deliver therapy as early as possible. A large scale amplifications by multiplex PCR of viral DNA could lower the cost and time for viral diagnosis. However, one has to consider various problems concerning PCR conditions such as annealing temperature of the forward and reverse primers of each virus, and avoidance of the self-annealing of primers and the size overlapping of PCR products for exact base calling. Thus, sensitive quadruplex PCR can be achieved by optimizing parameters such as primers, 1.5mM magnesium and 200 uM dNTPs concentrations. The concentration of HSV-1, HSV-2, CMV and EBV primers were 2.0 pmoles, 1.5 pmoles, 1.0 pmoles and 1.0 pmoles, respectively. Optimal annealing temperature was 54℃. And by this method we could detect 10 copies of reconstructed template plasmid DNA which were cloned to vectors containing target sequences of viral DNA, PCR products of HSV-1 having 271 bp, 231 bp for HSV-2, 368 bp for CMV, and 326 bp for EBV were separated on 2.0% agarose gel electrophoresis and confirmed by direct sequencing, The present study showed that quadruplex PCR assay described herein has a potentiality for application in clinical diagnosis, when a rapid and accurate detection, and typing of viruses HSV-1, HSV-2, CMV or EBV are necessary.
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