Development And Clinical Application Of SARS Virus CDNA Restriction Endoenzyme Analysis Kit

Sever actue respiratory syndrome emerged in southern China in late 2002 and rapidly spread to different areas of the Far East as well as several countries around the globe. When the disease's ability to spread to distant areas within a very short period of time became obvious.This diseas ofen onset quickly.This devastating disease has a relatively high mortality. When the outbreak of this apparently novel infectious disease termed severe acute respiratory syndrome (SARS ) came to an end in July 2003,it had caused over 7761 probable cases worldwide and more than 623 deaths.SARS is a new emerging viral disease.A novel coronavirus(SARS Cov) was isolated from patients with SARS.SARS Cov was revealed to have a 30kb long viral genome.Containing 14 potential open reading frames(ORFs).The sequence of SARS Cov reveals the prensence of ORFs for four structural proteins. i.e. the spike,membrane,envelope and nucleocapsid protein.The clinical case definition of SARS is essentially one of contact with other patients with SARS.It is not easy to differentiate SARS from other causes of pneumoniaWhen SARS had been transmitted all over the word.It disturbed people's normal work and vital order. How to give a right,quickly and effective clinical diagnosis and theraputics is a important problem.Correcting clinical diagnosis,especially,earier diagnosis is the key to the control of this disease. We can have diagnosis on people who have clinical symptom and objective sign ofatype pneumonia patient,imitate contactor,clinical science observer and doubtful aptype pneumonia by scientific detection.in this situation,we designde a new diagnosis and use it into early detection.lt is not only give diagnosis on cotactor,but it also can give a monitoring on the contaminated water source and food.First, we use GCG software to analyze SARS coronavirus genome's conserved fragment and eliminate the familiar fragment with human,bacteria and virus. Make the primers have its specifity.lt ensure the accuration of clinical diagnosis.Secondly,we alternate S protein nucleinic acid conserved fragment to design primer.we diterminate a resitriction endoenzyme sit of Sal-1 .We analyze the mutated SARS Cov all over the word and find out there is no mutation in Sal-1 restriction endoenzyme sit in S-cDNA fragment.lt is the key to ensure the accurat rate of our diagnosis kit.l.The isolation of SARS CovWe use Trizol to isolate SARS Cov and abstract SARS RNA.lt plays an important role to our later work. 2. Ascertainment of S-cDNAWe use GCG software to analyze SARS coronavirus genome's conserved fragment and eliminate the familiar fragment with human,bacteria and virus. Make the primers have its specifity.lt ensure the accuration of clinical diagnosis.we alternate a nucleic acid fragmen with 806bp.3.CloneofS-cDNAWe conjunct S-cDNA fragment into pGEM-T vetonUsing promega company's reactant kit.We use high voltage electroporation to transfer plasmid into competent cell.We employed X-gal/IPTG flat plate to blot passive clone strain.Obtaining passive clone strain successfully.4.Authentication of passive cloneWe design 3 programs to prob passive clone strain. a.DNA sequence analysisWe employ passive clone's PCR production and analyze cDNA sequence.lt indicate that the fragment which we inserted is right. b.Nested-PCR detectionWe use SST-l/SST-2^ SS-l/SS-2, SS-l/SST-2. SST-l/SS-2 to do nested-PCR. Agarose gel electrophoresis analyze consequence is coincidence with our anticipate.We obtained 230bp^ 305bp^ 711bp^ 806bp strips.Our objective DNA is right. c.Sal- I restriction endoenzyme analysis and authenticationWe use Sal-1 to analyze cDNA. Agarose gel electrophoresis analyze consequence indicate that the inserted S-cDNA is the correct one. 5.Bolting of specific Sal-1 restriction endoenzyme sit.The right Sal-1 restriction endoenzyme sit is key to accurate rate of this reactant kit.Restriction endoenzyme analysis result indicate that there are 463bp and 343bp two strips.lt is a key technique of this reactant kitRestriction endoenzyme is a gene allocate instrumentThis reactant kit is applyed to diagnose virus.This detected process give a new guidance to the detection of viral disease and other disease.This study compared with traditional diagnosis have high sensitivity and avoid unspecifity.It inherit good qulity of traditional detected method.