31 results about "Nested rt pcr" patented technology

The invention discloses a preparation method for a monoclonal antibody. According to the method, magnetic granular microballoon spheres of an immobilized antigen are added into B lymphocyte culture micropores, magnetic separation is carried out on the magnetic granular microballoon spheres so as to allow the spheres to enter into corresponding microplates, a single B lymphocyte which secretes a specific antibody is detected by using the method of immunochemiluminescence or immunofluorescence, a light chain variable region gene and a heavy chain variable region gene of the antibody are amplified by using the method of single-cell RT-PCR and nested RT-PCR and are recombined to expression plasmid containing a constant domain of the antibody, and a host cell is transfected to express the monoclonal antibody.

The invention belongs to the technical field of gene engineering, and discloses a primer combination for detecting leukaemia fusion genes, a kit containing the primer combination, and a multiplex nested RT-PCR (reverse transcription-polymerase chain reaction) method for performing leukaemia fusion gene detection by using the primer combination or kit. The method is based on a multiplex nested RT-PCR technique, and thus, is simple and quick and has high sensitivity. Besides, the reasonable primer combination is utilized to effectively avoid interactions among multiple primer pairs, thereby reducing the detection errors. The detection method can be utilized to comprehensively perform qualitative detection on 43 leukaemia fusion genes, thereby saving the reagent consumption and lowering the detection cost. The detection method has wide detection range, and is suitable for detecting mass samples in clinic.

The present invention provides nested RT-PCR primers, a detection method and a kit used for detecting porcine deltacoronavirus. The nested RT-PCR primers are two pairs of designed primers according toa highly conserved specific sequence of the porcine deltacoronavirus. The detection method comprises the following steps: sample RNA is extracted; the obtained sample RNA is used as a template, and the primers are used to conduct an RT-PCR reaction; and a reaction product is subjected to an agarose gel electrophoresis analysis. The kit comprises the primers, an amplification reagent, a positive control and a negative control. A provided technical scheme is strong in specificity, high in sensitivity, good in an anti-interference performance and also strong in reliability, overcomes problems oflow sensitivity, poor specificity, etc. of common detection methods, and is relatively low in costs, short in detection cycles and strong in practicability compared with existing detection methods ofnucleic acid hybridization, gene chips, etc.

The invention relates to an RT-PCR detection method of a south rice black-streaked dwarf virus (SRBSDV). In the detection method, SRBSDV specific primers are used for carrying out RT-PCR amplification on a sample to be detected and then carrying out electrophoretic identification. The specific primers are obtained by the method that by utilizing the conservatism of the evolution of viral nucleotide sequences, the tenth section of RNA of SRBSDV and nucleotide sequences of the tenth section of RNA of similar viruses are searched and downloaded from NCBI / GenBank, the sequences are compared by utilizing MEGA4.0 software, and then a pair of primers which are shared by all the SRBSDV nucleotide sequences reported by different researchers and are different from other viral sequences are found out from a file with an extension name of mas. Proved by a BLAST result, sequences which 100 percent cover the primers and 100 percent same as the primers are all the SRBSDV nucleotide sequences. The primers can be used for accurately separating SRBSDV diseased plants from ordinary RBSDV diseased plants without a nested RT-PCR detection.

The invention discloses a nested RT-PCR detection method for differentiating variant strains and classic strains of PEDV, belonging to the technical field of rapid detection of PEDV virus. According to the nested RT-PCR detection method, the disadvantages that the steps of isolation, identification and gene sequencing of viruses are tedious, the cost is high, and the consumed time is long are overcome, and the problems of low specificity and poor sensitivity of the traditional RT-PCR are solved. The nested RT-PCR detection method has the beneficial effects that the operation is simple and convenient, the use is simple, the accuracy is high, the specificity is strong, and the sensitivity is high; the method is capable of detecting whether PEDV on infected animal engine bodies is the variant strain and qualitatively detecting whether PEDV exists in epidemic materials, so that the detection technique for differentiating whether PEDV is the variant strain in the production is greatly facilitated, and the PEDV research contents in the laboratory are enriched.

The invention discloses a nested RT-PCR method and a primer combination for detecting potato mop-top virus. The method comprises the following steps: firstly, conducting reverse transcription by virtue of a random primer by taking RNA as a template, so as to obtain cDNA; secondly, taking the cDNA as a template; and thirdly, conducting the second turn of PCR by taking the fragment 1 of the first PCR amplified product as a template, wherein by virtue of two turns of the PCR reactions, the specificity and the sensitivity of detecting and identifying the virus are improved. Therefore, the nested RT-PCR method, when used for detecting the PMTV, is high in virus detection sensitivity and strong in specificity.

The invention provides two primer pairs. One primer pair includes nucleotide sequences shown as SEQ ID NO:1 and SEQ ID NO:2, and the other primer pair includes nucleotide sequences shown as SEQ ID NO:3 and SEQ ID NO:4. The invention further provides application of the two primer pairs in preparing products for detecting the Zika virus. The invention further provides a reagent kit comprising the two primer pairs and application. As proved by experiments, the two primer pairs are good in specificity and repeatability and high in sensitivity, and a Zika virus detecting method established based on the two primer pairs can be used for sensitively, accurately, stably and rapidly detecting whether the Zika virus exists or not and has great significance on clinical rapid diagnosis and public prevention and control over the Zika virus in China.

The invention relates to a nested RT-PCR (reverse transcription-polymerase chain reaction) primer group, a detection method and a kit for CYVCV (Citrus Yellow Vein Clearing Virus) and belongs to the technical field of molecular biology. The invention provides nested RT-PCR external nest primer pairs CYVCV-1f and CYVCV-1r and nested RT-PCR internal nest primer pairs CYVCV-2f and CYVCV-2r for CYVCV. The external nest primer pairs are utilized to carry out a first round of RT-PCR amplification; then a product obtained in the first round of RT-PCR amplification is used as a template and the internal nest primer pairs are utilized to carry out a second round of RT-PCR amplification; a PCR product obtained in the second round is detected to obtain a sample to be detected of the 469bp PCR product, i.e. the sample infected by CYVCV. The nested RT-PCR primer group is simple and convenient to operate and has good repeatability and high accuracy; sensitivity of the nested RT-PCR primer group is 100 times of that of common RT-PCR; and the nested RT-PCR primer group also can timely detect viruses in the incubation period.

A hog cholera virus nested RT-PCR detection kit is provided. A hog cholera virus PCR monitoring method is established on the basis of CSFV hog cholera wild virus strain complete genome sequence HEBZ (GU592790) included in Gen Bank as a reference, wherein amplified target fragment is 546 nt. The method is excellent in specificity, sensitivity and repeatability.

The invention discloses a detection method for EV71 virus in an environmental water body, wherein a semi-nested RT-PCR (Reverse Transcription-Polymerase Chain Reaction) and real-time fluorescence quantification RT-PCR reaction system is established to detect EV71 virus in the environmental water body by using a recombinant plasmid as a reference product, and the recombinant plasmid is prepared by using a primer and a probe aiming at EV71 virus designed according to a genetic conservative fragment of enterovirus belonging to a VP1 serotype area. The method has good specificity and high sensitivity and precision, and can qualitatively and quantitatively detect EV71 virus in the environmental water body accurately.

The invention provides a semi-nested RT-PCR detection method of canine astrovirus. The method comprises the following steps: 1, designing a primer with the conservative fragment of canine astrovirus gene as a target; 2, processing a detection sample, and extracting total RNA; 3, reversely transcribing the total RNA into cDNA; and 4, detecting the canine astrovirus in the sample through the semi-nested RT-PCR method. The method has the characteristics of high sensitivity, good specificity, simplicity, fastness and the like, and is suitable for the pathogenic diagnosis and the epidemiological studies of the canine astrovirus.

The invention discloses a detection method for CVA16 in an environmental water body, wherein a semi-nested RT-PCR (Reverse Transcription-Polymerase Chain Reaction) and real-time fluorescence quantification RT-PCR reaction system is established to detect CVA16 virus in the environmental water body by using a recombinant plasmid as a reference product, and the recombinant plasmid is prepared by using a primer and a probe aiming at CVA16 virus designed according to a genetic conservative fragment of enterovirus belonging to a VP1 serotype area. The method has good specificity and high sensitivity and precision, and can qualitatively and quantitatively detect CVA16 virus in the environmental water body accurately.

The invention belongs to the technical field of biological detection, in particular relates to semi-nested reverse transcription-polymerase chain reaction (RT-PCR) typing primers and a kit for identifying different serotypes of live vaccines for infectious bronchitis virus (IBV). The typing detection primers provided by the invention comprise universal primers having sequences shown in SEQ ID 1-2,and serotype specific primers having sequences shown in SEQ ID 3-6. The invention also discloses the kit comprising the typing detection primers. The M-type, 4 / 91-type, LDT3-A-type and QX-type serotype live vaccines for the IBV are identified by utilizing the primers and the kit which are provided by the invention and by adopting a semi-nested RT-PCR method; compared with the conventional PCR methods, the semi-nested RT-PCR method has stronger specificity and sensitivity; the method also has the characteristics of being fast, high in efficiency and low in cost, can complete the sample detection within 5-6h, and overcomes the defect of longer time consumption of the traditional serum neutralization test method; furthermore, the method is suitable for the detection and analysis a great batch of samples.

The invention discloses an infectious spleen and kidney necrosis virus (ISKNV) gene intron and its use in distinguishing of live and inactivated ISKNVs and belongs to the field of virus detection. The intron is located on ORF003L of an ISKNV genome NC_003494.1, has length of 80bp and is named as IN-3. The invention discloses the intron of ISKNV. The intron is named as IN-3. Through the characteristics of transcription shearing of the intron IN-3, the intron IN-3 is used as a virus complete inactivation index, and an ISKNV inactivation rapid detection nested RT-PCR method is established. The ISKNV gene intron can shorten an inactivation test cycle in ISKNV cell inactivated vaccine production process and improve vaccine production efficiency.

The invention relates to the technical field of biology, in particular to a primer group, a kit with the primer and application. The primer group comprises an outer nested primer and an inner nested primer; a sequence of a forward primer of the outer nested primer is shown as SEQ ID NO.1; a sequence of a reverse primer of the outer nested primer is shown as SEQ ID NO.2; a sequence of a forward primer of the inner nested primer is shown as SEQ ID NO.3; a sequence of a reverse primer of the inner nested primer is shown as SEQ ID NO.4. The invention further provides a foot and mouth disease virusamplification method. A nested RT-PCR (RT-nPCR) method is quick and high in sensitivity, stability and specificity, and simultaneous amplification of O-type, A-type and AsiaI-type foot and mouth disease viruses can be realized.

The invention discloses a nested RT-PCR(reverse transcription-polymerase chain reaction)primer, a kit containing the primer and a detection method, which are used for detecting porcine Kobuvirus. The nested RT-PCR primer comprises (1) outward primers P1 and P2, the sequences of which are respectively as shown in SEQ ID NO.1 and SEQ ID NO.2, and (2) inward primers P3 and P4, the sequences of which are respectively as shown in SEQ ID NO.3 and SEQ ID NO.4. The detection method comprises the steps of: (1) extracting the total RNA (Ribose Nucleic Acid) of a sample; (2) obtaining cDNA (complementary deoxyribonucleic acid) by taking the total RNA as a template through RT (Reverse Transcription); and (3) detecting by taking the cDNA as a template after carrying out nested PCR (Polymerase Chain Reaction). The detection method has the advantages of good specificity, high sensitivity and simplicity in operation; and the kit has the advantages of low cost, fast detection speed, difficulty in pollution, easiness in determination of results and suitability for diagnosis of porcine Kobuvirus infection and very good application prospect.

The invention relates to a primer set and kit for detecting ZIKV based on one-step fluorescent quantitative nested RT-PCR, belonging to the field of biotechnology. The primer set comprises a pair of external primers and a pair of internal primers, wherein the upstream and downstream sequences of the external primers are as shown in SEQ ID No. 1-2, and the upstream and downstream sequences of the internal primers are as shown in SEQ ID No. 3-4. The one-step fluorescent quantitative nested RT-PCR primers for ZIKV designed in the invention have good specificity, high sensitivity and good repeatability. A designed one-step fluorescence quantitative nested RT-PCR detection method can sensitively, accurately, stably and rapidly detect the presence of ZIKV and is capable of detecting viruses withthe lowest concentration of 6.4*10<-1> TCID50 / mL. The one-step fluorescence quantitative nested RT-PCR detection method provides a technical means for rapid and sensitive detection of ZIKV, and is ofgreat significance to the rapid clinical diagnosis and public prevention and control of ZIKV at present.

The invention relates to a narcissus late season yellows virus detection kit and method specific for narcissus late season yellows virus detection. The knit comprises an outside upstream primer, an outside downstream primer, an inside upstream primer, an inside downstream primer, an RTBuffer, an RNA enzyme inhibiting factor, reverse transcriptase, dNTPs, a PCR Buffer, MgCl2, TaqDNA polymeras, positive control, negative control and RNase-free ddH2O. Specific primers are designed according to narcissus late season yellows virus gene sequences, and a nested RT-PCR technology is utilized to detect narcissus late season yellows viruses. Firstly, inverse transcription is carried out with extracted RNA as the template to synthesize cDNA, first-round PCR amplification is carried out with the cDNA as the template, second-round PCR amplification is then carried out with first-round PCR products as the template, and agarose gel electrophoresis detection is conducted on amplification products: if a specificity target fragment of the size of 539bp appear, the judgment result indicates that a detected virus is a narcissus late season yellows virus. The narcissus late season yellows virus detection kit and method have the advantages of being strong in specificity, high in sensitivity, good in accuracy and short in detection time and are suitable for quickly detecting and monitoring narcissus late season yellows viruses in entry ports, exit ports and agricultural production.

The invention discloses a reagent kit for detecting an H4 subtype avian influenza virus based on nested RT-PCR. The reagent kit comprises a primer pair set composed of an outer primer pair and an inner primer pair, wherein the outer primer pair is composed of two pieces of single-chain DNA shown in the sequence 1 and sequence 2 in a sequence table, and the inner primer pair is composed of two pieces of single-chain DNA shown in the sequence 3 and sequence 4 in the sequence table. It is proved through experiments that by means of the primer pair set provided in the reagent kit, the H4 subtype avian influenza virus can be obtained through specific amplification while other common poultry disease pathogens are not amplified, and the lower detection limit for the H4 subtype avian influenza virus is 360 fg / micro L. By means of the nested RY-PCR method established through the reagent kit, a quick, specific and sensitive detection method is provided for early diagnosis and effective prevention and treatment of the H4 subtype avian influenza virus.

The invention relates to a nest type RT-PCR detection kit and a detection method for narcissus degeneration virus, which are specially used for detecting narcissus degeneration virus. The kit comprises an outer-side forward primer, an outer-side reverse primer, an inner-side forward primer, an inner-side reverse primer, an RT Buffer, an RNA enzyme inhibiting factor, a reverse transcriptase, dNTPs, a PCR Buffer, MgCl2, Taq DNA polymerase, a positive control, a negative control and RNase-free ddH2O. The nest type RT-PCR detection kit and the detection method conduct detection to the narcissus degeneration virus by adopting nest type RT-PCR technology, and have the advantages of being strong in specificity, high in sensitivity, simple and convenient to operate, and fast in detection, and are applicable to fast detection, epidemic situation monitoring and early-warning and forecasting on the narcissus degeneration virus in China export and import quarantine and agricultural production, thus being wide in application prospects.

The invention discloses a detection method for CVA16 in an environmental water body, wherein a semi-nested RT-PCR (Reverse Transcription-Polymerase Chain Reaction) and real-time fluorescence quantification RT-PCR reaction system is established to detect CVA16 virus in the environmental water body by using a recombinant plasmid as a reference product, and the recombinant plasmid is prepared by using a primer and a probe aiming at CVA16 virus designed according to a genetic conservative fragment of enterovirus belonging to a VP1 serotype area. The method has good specificity and high sensitivity and precision, and can qualitatively and quantitatively detect CVA16 virus in the environmental water body accurately.

The invention discloses a primer combination for simultaneous identification of three porcine epidemic diarrhea virus strains. The primer combination is composed of 4 specific primers: an upstream primer F1, an upstream primer F2, an upstream primer F3 and a downstream primer UR respectively. The invention also provides a general semi-nested RT-PCR method for simultaneous identification of three porcine epidemic diarrhea viruses with the primer combination. According to the primer combination and the general semi-nested RT-PCR method for simultaneous identification of three porcine epidemic diarrhea virus strains provided by the invention, all the three porcine epidemic diarrhea virus strains that are already reported and appear can be identified and distinguished simultaneously in one test by one method. The detection method is established for the first time in the world to be able to detect three different genotype porcine epidemic diarrhea virus prevalent strains at the same time, and provides a new and effective detection method for identification and clinical diagnosis of the viruses in the future.

The invention belongs to the technical field of gene engineering, and discloses a primer combination for detecting leukaemia fusion genes, a kit containing the primer combination, and a multiplex nested RT-PCR (reverse transcription-polymerase chain reaction) method for performing leukaemia fusion gene detection by using the primer combination or kit. The method is based on a multiplex nested RT-PCR technique, and thus, is simple and quick and has high sensitivity. Besides, the reasonable primer combination is utilized to effectively avoid interactions among multiple primer pairs, thereby reducing the detection errors. The detection method can be utilized to comprehensively perform qualitative detection on 43 leukaemia fusion genes, thereby saving the reagent consumption and lowering the detection cost. The detection method has wide detection range, and is suitable for detecting mass samples in clinic.

The invention discloses a nested RT-PCR(reverse transcription-polymerase chain reaction)primer, a kit containing the primer and a detection method, which are used for detecting porcine Kobuvirus. The nested RT-PCR primer comprises (1) outward primers P1 and P2, the sequences of which are respectively as shown in SEQ ID NO.1 and SEQ ID NO.2, and (2) inward primers P3 and P4, the sequences of which are respectively as shown in SEQ ID NO.3 and SEQ ID NO.4. The detection method comprises the steps of: (1) extracting the total RNA (Ribose Nucleic Acid) of a sample; (2) obtaining cDNA (complementary deoxyribonucleic acid) by taking the total RNA as a template through RT (Reverse Transcription); and (3) detecting by taking the cDNA as a template after carrying out nested PCR (Polymerase Chain Reaction). The detection method has the advantages of good specificity, high sensitivity and simplicity in operation; and the kit has the advantages of low cost, fast detection speed, difficulty in pollution, easiness in determination of results and suitability for diagnosis of porcine Kobuvirus infection and very good application prospect.

The invention discloses a detection method for CVA10 in an environmental water body, wherein a semi-nested RT-PCR (Reverse Transcription-Polymerase Chain Reaction) and real-time fluorescence quantification RT-PCR reaction system is established to detect CVA10 virus in the environmental water body by using a recombinant plasmid as a reference product, and the recombinant plasmid is prepared by using a primer and a probe aiming at CVA10 virus designed according to a genetic conservative fragment of enterovirus belonging to a VP1 serotype area. The method has good specificity and high sensitivity and precision, and can qualitatively and quantitatively detect CVA10 virus in the environmental water body accurately.

The invention relates to a narcissus late season yellows virus detection kit and method specific for narcissus late season yellows virus detection. The knit comprises an outside upstream primer, an outside downstream primer, an inside upstream primer, an inside downstream primer, an RTBuffer, an RNA enzyme inhibiting factor, reverse transcriptase, dNTPs, a PCR Buffer, MgCl2, TaqDNA polymeras, positive control, negative control and RNase-free ddH2O. Specific primers are designed according to narcissus late season yellows virus gene sequences, and a nested RT-PCR technology is utilized to detect narcissus late season yellows viruses. Firstly, inverse transcription is carried out with extracted RNA as the template to synthesize cDNA, first-round PCR amplification is carried out with the cDNA as the template, second-round PCR amplification is then carried out with first-round PCR products as the template, and agarose gel electrophoresis detection is conducted on amplification products: if a specificity target fragment of the size of 539bp appear, the judgment result indicates that a detected virus is a narcissus late season yellows virus. The narcissus late season yellows virus detection kit and method have the advantages of being strong in specificity, high in sensitivity, good in accuracy and short in detection time and are suitable for quickly detecting and monitoring narcissus late season yellows viruses in entry ports, exit ports and agricultural production.