57 results about "Orf2 gene" patented technology

The invention discloses porcine circovirus II-type recombinant baculovirus as well as a preparation method and application thereof. ORF2 gene is artificially synthesized by referring to a PCV2b isolated strain ORF2 gene sequence; the synthesized ORF2 gene is connected to pFBDPHmHNM1P10eGFP plasmid by adopting the plasmid as a framework vector, so that a baculovirus transfer vector pFBDPHm 30RF2 is obtained. The baculovirus transfer vector pFBDPHm30RF2 is mixed with DH10Bac escherichia coli competent cells, and the positive bacterial colony is selected to obtain a recombinant rod granule rBac-PVR30RF2; the rod granule is transferred with a sf9 cell to obtain the recombinant baculovirus QP-Ac-30RF2. The recombinant baculovirus can be used for efficiently expressing the PCV20RF2 protein and forming virus-like particles. The VLP which is expressed and packaged by the recombinant baculovirus disclosed by the invention is used for preparing inactivated vaccine, and the organism is induced to generate specific immunity response after a 28-day-aged piglet is immunized, and the pig body can be completely protected from virulent attacks of the porcine circovirus.

The invention mainly aims to provide a porcine circovirus type 2 (PCV2) subunit vaccine and a preparation method thereof. Particularly, a baculovirus vector expression system is utilized to express a large amount of recombinant open reading frame type 2 (ORF2) protein in insect cells, so that the subunit vaccine with good immunity effect is developed. A bac-to-bac baculovirus expression system is adopted to perform whole gene amplification on the porcine circovirus type 2 ORF2 gene, and a melittin signal peptide nucleotide sequence is introduced into a terminal 5', so that the recombinant baculovirus of an open reading frame containing the melittin signal peptide nucleotide sequence and a PVC2ORF2 gene sequence is established, wherein the infected insect cell expresses the recombinant ORF2 protein with efficient and high PCV2 antigenicity; and thus the subunit vaccine containing the PCV2 recombinant ORF2 protein is prepared. The inoculation experiments of piglets show that the subunit vaccine has a good immunity protection effect.

The invention relates to porcine circovirus PCV2 ORF2 gene optimized by using escherichia coli expression codon and a preparation method of PCV2 ORF2 capsid protein virus-like particles derived from the escherichia coli.

The invention discloses a preparation method of a recombinant porcine circovirus type 2 Cap antigen, which comprises the following steps: using a yeast expression system to amplify the PCV2-ORF2 gene; then, constructing an expression engineering bacterium: inserting an amplification sequence into a yeast expression vector to form a recombinant expression vector, linearizing, and transforming into a pichia pastoris host cell so as to finish the construction of the expression engineering bacterium; and finally, carrying out secretory expression on the recombinant microzyme to obtain the recombinant porcine circovirus type 2 Cap antigen protein. The yeast expression system can carry out processing, folding and posttranslational modification on the expressed protein, so that the expressed protein has biological activity. Compared with Escherichia coli, the yeast expression system has more advantages; and compared with a mammal cell expression system, the yeast expression system is more convenient to operate. Compared with Saccharomyces cerevisiae, the pichia pastoris can not be easily subjected to hyperglycosylation, thereby facilitating the separation and purification steps. By using the yeast expression system, the invention has the advantages of simple operation process, high expression quantity and low production cost, can implement large-scale production, and is beneficial to the popularization and application of the porcine circovirus subunit vaccine.

The invention relates to a porcin circovirus type 2 (PCV2) recombinant autographa californica multicapsid nucleopolyhedrovirus construction method and a subunit vaccine preparation method thereof. The novel recombinant lucerne fork vein noctuid nuclear polyhedrosis virus construction method comprises the steps that: the main surface antigen which expresses the PCV2 is used to prepare the subunit vaccine with better immunity. Specifically, in the carrier expression system of the PCV2 recombinant autographa californica multicapsid nucleopolyhedrovirus, a hepatitis E virus open reading frame (ORF2) gene of the PCV2 is directionally inserted, so the gene efficiently expresses in insect cells and has good immunogenicity, the prepared corresponding subunit vaccine can stimulate pigs to generate the protective immunoreaction against the attack of the strong virus of the PCV2.

The invention discloses a series of N-terminal truncated PCV2-type ORF2 (Open Reading Frame 2) genes, a virus-like particle of the ORF2 gene and a preparation method of the virus-like particle, a vaccine including the truncated virus-like particle and an application of the vaccine in the prevention of post-weaning multisystemic wasting syndrome infection. The virus-like particle assembled by truncated PCV-ORF2 genes disclosed by the invention has good immunogenicity and protection. Since no denaturing agent is used in the preparation and purification process disclosed by the invention, the activity of the target protein is protected to the greatest extent.

The invention discloses an expression vector of a Cap protein of a porcine circovirus (PCV) 2 as well as a construction method and application thereof. The expression vector is obtained by cloning an optimized codon obtained after optimization of a gene ORF2 of the PCV 2 in a pET-28a vector. The expression vector and the construction method have the beneficial effects that the prokaryotic expression vector capable of efficiently expressing the main immunogenic Cap protein of the PCV 2 can be constructed and the Cap protein of the PCV 2 is efficiently expressed by utilizing escherichia coli, thus obviously reducing the antigen production cost, simplifying the production process and providing the cheap and fine vaccine product for the pig industry.

The invention discloses a construction method and an application of a porcine encephalomyocarditis virus BD2 strain full-length infectious clone. On the basis of the constructed porcine encephalomyocarditis virus BD2 strain full-length infectious clone, an ORF2 gene of porcine circovirus is interpolated into an L gene encoding virus pilot protein, so that the encephalomyocarditis virus BD2 strain full-length infectious clone, which stably carries the ORF2 gene of the porcine circovirus 2, is successfully constructed. Both the constructed EMCVBD2 strain full-length infectious clone and the encephalomyocarditis virus BD2 strain full-length infectious clone, which stably carries the ORF2 gene of the porcine circovirus 2, can rescue infectious recombinant virus; therefore, the EMCVBD2 strain full-length infectious clone and the encephalomyocarditis virus BD2 strain full-length infectious clone, which stably carries the ORF2 gene of the porcine circovirus 2, can be applied to vaccine preparation and virus researches.

A chimeric hog cyclovirus PCV1-2 is disclosed. Its configuring method include ssuch steps as using the PVC2-ORF2 of hog cyclovirus PVC2 to replace the PVC1-ORF2 of PVC1, configuring a fragment of deficiency PCV1-ORF2 genome (pSK-PVC1 delta ORF2), and linking it to a fragment of PCV2-ORF2 genome. It can be used to prevent and treat the multi system failure syndrome of delactated piglet by PCV1-2 infectious immunizing cesarean section and deprivating foremilk.

The invention provides a restructing adenovirus of 2-type pig-ring virus and constructing method thereof, and the preventing appliances of pigs' relevant diseases caused by PCV2, including: augment and clone of PCV2 ORF2 genes; construction of recombinant shuttle vectors containing ORF2 genes; construction of recombinant adenovirus plasmid vectors; acquirement of recombinant adenovirus. Said recombinant adenovirus could determinately grow inside the bodies and continueously express ORF2 proteins to stimulate the organisms which generate neutralizing antibodies for ORF2 proteins, and offer endurance protection to pigs with promising future in the prevention and cure of PCV2, which could prevent efficiently poor quality of male-pig sperms, abortion, dead fetus, mummy and low production of female-pigs to increase the uniformity of pigs greatly.

The invention discloses a RT-RPA-lateral flow tomography kit for doubly detecting hepatitis e virus and hepatitis a virus. The kit comprises an upstream primer, an intermediate probe and a downstreamprimer which are applicable to hepatitis e virus ORF2 gene sequence in the RT-RAP amplification technology, and / or an upstream primer, an intermediate probe and a downstream primer which are applicable to hepatitis a virus VP1 gene sequence, general agents for recombinase polymerase amplification technology, and a lateral flow tomography test strip, wherein the lateral flow tomography test stripcomprises a sample adding pad, a control line, a #1 detecting line and / or a #2 detecting line; the control line, the detecting lines and the primers are combined with a probe label. With the adoptionof the kit, the hepatitis e virus ORF2 gene and / or hepatitis a virus VP1 gene in a sample can be rapidly, sensitively and specifically detected on site in a non-lab environment.

The invention relates to a fourfold real-time fluorescent PCR identifying and detecting method for porcine circoviruses 1 to 4, and belongs to the technical field of biology. In accordance with ORF1 and ORF2 genes of different genotypes of PCV, specific primers and probe sequences, capable of identifying PCV1, PCV2, PCV3 and PCV4, and a probe of the PCV1-4 strains labeled with different fluorescein can be designed; each probe can be labeled with any fluorescein, and fluorescein for labeling the four probes needs to be detected by different detection passages; the primers and probes for detecting the PCV1-4 strains, or the primer and probe for identifying the PCV1-4 strains are placed in the same reaction tube, a fluorescent PCR amplification instrument is used for a real-time fluorescent PCR reaction, and the same sample is detected once, so that identification and detection on the PCV 1-4 strains can be completed.

The invention discloses a recombinant porcine pseudorabies virus (Herpesviridae) strain used for expression of porcine circovirus type II ORF2 gene. Preservation number of the recombinant porcine pseudorabies virus strain is CCTCC No.V201315. According to the preparation method, PCV2ORF2 gene is inserted into common carrier PG so as to construct transferring plasmid PGO; monolayer ST cells are inoculated with PRVTK<-> / gG<-> / gE<-> virus by 2h of adsorption; ST cells are transfected with plasmid PGO, wherein fusion degree of the monolayer ST cells is 80 to 90%; the recombinant porcine pseudorabies virus PGO strain is obtained by plaque purification, and is used for immunization of mouse. It is shown by results that commercial PCV2 inactivated vaccine and a group immunized by the recombinant porcine pseudorabies virus PGO strain are both capable of inducing specific humoral immune response of mouse, antibody titers of both the commercial PCV2 inactivated vaccine and the group are obviously higher than that of a group immunized by DMEM medium, and difference is significant (p<0.05). It is shown by the results of mouse lymphocyte proliferation test that specific ceullar immune response caused by the group immunized by the recombinant porcine pseudorabies virus PGO strain is more obvious than that caused by the commercial PCV2 inactivated vaccine and the group immunized by DMEM medium, and difference is obvious (p<0.05). In addition, the group immunized by the recombinant porcine pseudorabies virus PGO strain is capable of resisting severe attack by PCV2. Therefore application of the recombinant porcine pseudorabies virus strain in development of a novel PCV2 vaccine is possible.

The technical scheme of the present invention is: a kind of porcine circovirus type II nucleic acid vaccine, respectively inserting porcine circovirus type II LY strain ORF2 and porcine IL-18 gene into two multiple cloning sites of the eukaryotic expression vector pIRES, The nucleic acid vaccine co-expresses porcine circovirus type II LY strain Cap and porcine IL-18 protein. The present invention is identified by PCR, double enzyme digestion and sequencing, which proves that the co-expression plasmid pIRES-ORF2 / PIL18 has been successfully constructed. The successfully constructed recombinant plasmid pIRES-ORF2 / PIL18 was transfected into porcine kidney PK-15 cells by liposome method, and the expression of the target protein was detected by RT-PCR and indirect immunofluorescence. The transcription of the two target genes was detected by RT-PCR; the specific bright green fluorescence of ORF2 protein was observed in the cytoplasm and nucleus by indirect immunofluorescence test, which proved that the protein was expressed.

The invention relates to a method for purifying recombinant porcine circovirus 2 type Cap protein. The recombinant porcine circovirus 2 type Cap protein is composed of ELP type elastin polypeptide genes and PCV-ORF2 genes. A preparation method of the purifying recombinant porcine circovirus 2 type Cap protein comprises the steps of expression vector construction and fusion protein expressing and purifying. Compared with an existing porcine circovirus vaccine purifying method, the method for purifying the recombinant porcine circovirus 2 type Cap protein has the advantages that preparation is easy and convenient, purifying is easy, and the price is low, and is suitable for large-scale production operation, and a foundation is provided for preparation, purification and application of subsequent recombinant porcine circovirus subunit vaccines.

The present invention relates to a PRRSV and PCV-2 bivalent recombinant fowlpox live vector vaccine, which pertains to the field of biotechnology. The present invention aims at providing the PRRSV and PCV-2 bivalent recombinant fowlpox live vector vaccine which has ORF5, ORF3 genes to express the structural glycoprotein of the PRRSV and ORF2 gene to express PCV-2 nucleocapsid protein and can be used as the live vector vaccine for the prevention of PRRS and PCV-2 infection in our country. The present invention constructs the pMD18T-ORF2, pMD18T-ORF5 and pMD18T-ORF3 plasmids, the plasmids are inserted to the downstream of the compound promoter ATI-P7.5 multiplied by 20 of the fowlpox virus expression vector pUTAL, at the same time, the ORF2 gene of the PCV2 are inserted to the downstream of the linking promoter P7.5 multiplied by 16, and the present invention further constructs the recombinant fowlpox virus gene transfer plasmid pUTAL-ORF2-ORF5-ORF3 which contains the ORF3-ORF3 gene of the PRRSV and the ORF2 gene of the PCV2. The present invention can be used as the live vector vaccine for the prevention of PRRS-PCV2 in our country.

The invention discloses a loop-mediated isothermal amplification (LAMP) diagnostic kit for porcine circovirus type 2. The diagnostic kit comprises 250muL of LAMPmix buffer solution, 20muL of (i)Bst( / i) deoxyribonucleic acid (DNA) polymerase, 150muL of ultrapure water, 40muL of mixed inner primer 1 and inner primer 2, of which the concentration is 40pmol / L, 40muL of mixed outer primer 1 and outer primer 2, of which the concentration is 5pmol / L, 20muL of negative control and 20muL of positive control. Two pairs of primers are designed and synthetized according to a porcine circovirus type 2(PCV-2) ORF2 gene sequence in GenBank, an LAMP diagnostic method for the porcine circovirus type 2 is built by optimization of the LAMP condition, and the sensitivity and specificity tests, and a simple and fast method is provided for rapid diagnosis of the porcine circovirus type 2 on the spot. Thus, quick quarantine needs of the basic level on the spot are met.

The present invention relates to a porcine circovirus 2 type recombinant subunit vaccine and a preparation method thereof. According to the present invention, an ORF1 gene dominant antigen epitope region and an ORF2 gene dominant antigen epitope region of porcine circovirus 2 type are reasonably combined; bioinformatics and a SOEing PCR technology are adopted to optimize codons to obtain an artificial gene sequence YSORF; the artificial gene sequence YSORF is constructed to a prokaryotic expression vector; escherichia coli is adopted to express recombinant fusion protein; and the resulting recombinant fusion protein is subjected to moderate purification, and then is mixed with an adjuvant to prepare a subunit vaccine containing an ORF1 protein fragment and an ORF2 protein fragment. With the present invention, the ORF1 gene dominant antigen epitope region and the ORF2 gene dominant antigen epitope region of the porcine circovirus 2 type are combined in a series connection manner creatively by using the flexible peptide, such that the number of the epitope is increased, and correct spatial conformation of the fusion protein is easily formed; the codons of the ORF1 and the ORF2 are optimized so as to increase an expression amount of the fusion protein and reduce production cost. The vaccine of the present invention has advantages of high yield, low cost, strong protection efficacy, and the like. In addition, the preparation method of the present invention is suitable for large-scale production.

This invention relates to modified ORF2 gene of porcine circovirus type 2 and its application. The modified ORF2 gene is obtained by: replacing the gene coding 41 amino acids at N-termal of natural ORF2 with the gene coding signal peptide of human tissue plasminogen activator, deleting terminator of ORF2 gene, and fusing the gene coding C-terminal of ORF2 with the gene coding the transmembrane peptide of avian influenza virus HA protein. The nucleotide sequence of ORF2 gene is shown in SEQ ID No.1. This invention also relates to eukaryotic plasmid pcDNA-spORF2 Delta41TM, and host E. coli strain DH5alpha / pcDNA-spORF2 Delta 41TM (CCTCC No.M207069). The ORF2 gene and the plasmid can be used for manufacturing the vaccine for preventing post-weaning multi-systematic syndrome.

The invention discloses a method for expressing an ORF2 protein of a porcine circovirus 3 (PCV3) by using Escherichia coli and application thereof. The method is as below: 1) cloning a solubility-enhancing tag GST gene, a solubility-enhancing tag His6-SUMO gene and a PVC3-ORF2 gene respectively; 2) ligating the obtained three genes to obtain a fusion gene; 3) ligating the obtained fusion gene to an E. coli expression vector to obtain a recombinant expression vector containing the fusion gene; 4) transforming the recombinant expression vector into E. coli expressing strain competent cells to obtain recombinant E. coli expressing strains containing the fusion gene; 5) culturing the obtained recombinant expression strains, and inducing expression of the fusion protein by IPTG; and 6) recovering the recombinant expression bacteria obtained by culture, breaking and purifying the recombinant expression bacteria to obtain the ORF2 protein of the porcine circovirus 3. The method not only obtains soluble protein with high expression, but also achieves high immunoreduction.

The invention relates to the field of biotechnology, in particular to a recombinant adenovirus for expressing porcine circovirus type-3 ORF2 gene and a preparation method and application of the recombinant adenovirus. The invention provides a recombinant adenovirus transfer plasmid containing a Cap protein coding gene of PCV3 and a recombinant adenovirus. According to the invention, through artificial codon optimization and signal peptide selection, high-level secretion expression of the Cap protein of PCV3 is realized, the structure and function of the obtained Cap protein of PCV3 are completely consistent with the natural state, the immunogenicity of the Cap protein is reserved to the greatest extent, and the Cap protein can be recognized by antigen presenting cells such as DC better and faster. The adenovirus vector vaccine provided by the invention has high immunogenicity and safety, can effectively activate the immune response of an organism, stimulates animals to generate high-level specific antibodies and neutralizing antibodies, and plays an excellent immune protection effect on porcine circovirus.

The invention discloses an LAMP visual quick detection kit for porcine circovirus type 2, and discloses an LAMP primer for detecting the porcine circovirus type 2 and a detection method thereof. Three pairs of primers are designed and synthesized according to the ORF2 gene sequence of the porcine circovirus type 2 (PCV2), an LAMP diagnosing method of the PCV 2 is established by optimizing the LAMP condition, a simple and quick method is provided to field quick diagnosis of the porcine circovirus type 2, and the LAMP visual quick detection kit for the PCV2 is prepared according to the method. The kit is simple in operation, and can directly determine the result of color changes to realize visualization, has easily determined reaction results, and can be easily popularized in clinical application.

The invention relates to a method for efficiently expressing an ORF2 (Open Reading Frame 2) gene of a goose astrovirus soluble capsid precursor, and application of the method, and belongs to the fieldof veterinary biological products. The ORF2 gene of the goose astrovirus is divided into two-segment expression according to the difference of gene sequence functions, and expression products are independently capsid precursor (short for cap) and capsid spike (short for spike). Two segments of proteins segmented according to functional zones can be expressed by escherichia coli, baculovirus and aCHO (Chinese Hamster Ovary) expression system. Through a purification method, the capsid precursor expression protein and the capsid spike expression protein of the goose astrovirus can be obtained.The expressed capsid precursor and capsid spike can be used for preparing a subunit vaccine and an egg yolk antibody of the goose astrovirus. The subunit vaccine and the egg yolk antibody prepared bythe method disclosed by the invention have a good immune effect and treatment effect, can be used for preventing diseases caused by the goose astrovirus and have the advantages of being in low in production cost, simple in operation and good in biological safety.

The invention relates to a recombinant virus of a PCV2 codon optimized ORF1 and ORF2 tandem gene, and belongs to the biopharmaceutical field. The artificially-synthesized codon optimized PCV2 ORF1 and ORF2 gene is named YHsfORF1-2. The YHsfORF1-2 is cloned to a baculovirus expression vector pFastBacHTA to obtain a recombinant plasmid pFBHYHsfORF1-2; and the recombinant plasmid is transfected to an Sf9 cell to obtain a recombinant baculovirus vFBHYHsfORF1-2. In the recombinant baculovirus vFBHYHsfORF1-2, ORF1 and ORF2 proteins can be successfully efficiently expressed, and can form virus-like particles. The invention also relates to an application of the recombinant virus in vaccines. Animal tests prove that inactivated vaccines prepared through using the recombinant virus can stimulate the body to generate efficient immune response and have a good immune protection effect on PCV2.

The invention discloses a porcine circovirus type III SYBR Green I real-time fluorescent quantitative PCR detection primer pair and a kit. The primer pair comprises a positive primer of nucleotide sequence as shown in SEQ ID NO:1 and a negative primer of nucleotide sequence as shown in SEQ ID NO:2. The primer pair is used for amplifying a partial segment of an ORF2 gene of the porcine circovirus type III, and the size of the amplified target gene segment is 131 bp. The invention also provides a porcine circovirus type III SYBR Green I real-time fluorescent quantitative PCR detection kit and a use method of the kit. When the primer pair is used for detecting the porcine circovirus type III, the primer pair has the characteristics of high specificity, high sensitivity, high repeatability, rapidness and the like, and can be applied to etiology detection of the porcine circovirus type III.

The invention discloses a PCV2 (porcine circovirus 2) recombinant baculovirus-like particle subunit vaccine and a preparation method thereof. According to the vaccine, an insect taken as a host cell is transfected with a eukaryotic expression vector containing a PCV2 recombinant baculovirus ORF2 gene fragment. The nucleotide sequence of the PCV2 recombinant baculovirus ORF2 gene fragment is as shown in SEQ ID NO. 1, and the eukaryotic expression vector is pAcGP67-A-PCV2. According to the method, the recombinant baculovirus containing the highly immunogenic PCV2 ORF2 nucleotide sequence is constructed by a molecular biology means, and PCV2 capsid protein can be efficiently expressed by the recombinant virus in the Sf9 cell line and assembled into PCV2 virus-like particles with PCV2 surfaceantigens in a cytoplasm. A harvested virus stock solution is processed by a filtration system and inactivated, and supplemented with an adjuvant, and the PCV2 recombinant baculovirus-like particle subunit vaccine with high immunity and good safety is prepared.

The invention relates to a chimeric virus-like particle vaccine and a preparation method therefor and application of the chimeric virus-like particle vaccine, in particular to a porcine Seneca valleyvirus and porcine circovirus-2 chimeric virus-like particle vaccine. According to the chimeric virus-like particle vaccine, sequences of VP0, VP3 and VP1 are connected in tandem for expression, wherein part of the VP3 sequences are replaced with porcine circovirus-2 ORF2 gene C-terminal epitope sequences to form a recombination sequence; the recombination sequence transfects sf9 cells after beingconstructed on recombinant bacmid for further expression to obtain porcine Seneca valley virus and porcine circovirus-2 chimeric virus-like particles. It is the first time for the invention to developthe porcine Seneca valley virus and porcine circovirus-2 chimeric virus-like particle vaccine by utilizing the chimeric virus-like particles, and the porcine Seneca valley virus and porcine circovirus-2 chimeric virus-like particle vaccine can exert a relatively good immune protection effect on the porcine Seneca valley virus and the porcine circovirus-2; and the vaccine of the invention is economical and practical, can effectively reduce the epidemic prevention cost of diseases, and provides a new method of preventing two diseases at the same time for breeding enterprises in China.

According to the fluorescent quantitative PCR detection method and kit for the porcine circovirus type II, corresponding specific primers and probes are designed for the ORF2 gene of the porcine circovirus type 2, namely the PCV2, cross reaction with PCV1, CSFV, PRRSV and PRV is avoided, and the detection limit and the quantitative limit are 10 copy numbers and 100 copy numbers respectively. The kit disclosed by the invention is high in sensitivity, strong in specificity and good in stability, a circulation threshold (Ct) and a standard DNA template have an excellent linear relationship in a concentration range of 102-106 copies / mu L, and a correlation coefficient is 1; the invention is simple in operation and good in repeatability, the variation coefficient of the Ct value in an intra-batch experiment ranges from 0.59% to 1.05%, the variation coefficient of the Ct value in an inter-batch experiment ranges from 1.9% to 4.2%, and a simple, convenient and rapid diagnosis method is provided for detection of the porcine circovirus type II and investigation of epidemiology.

The invention discloses application of recombinant adenovirus containing porcine circovirus type 2 ORF2 genes as a standard sample in nucleic acid amplification testing. The adenovirus recombination technology is used to prepare the recombinant adenovirus containing the porcine circovirus type 2 ORF2 genes, the recombinant adenovirus is used as the standard sample of the nucleic acid amplification testing (NAT), the standard sample is stable, simple to prepare and free of biosafety risks, virus particular structure of the recombinant adenovirus is generally similar to that of pig prokaryotic virus, whole-process quality control of virus DNA extraction and amplification detection can be achieved, features such as certain quantity value are achieved, and the recombinant adenovirus is suitable for the quality control of a porcine circovirus type 2 nucleic acid amplification detection method and evaluation of detection reagents and related abilities of laboratories.