31 results about "Nested rt pcr" patented technology

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 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 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 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 present invention aims to provide a nested RT-PCR method and the kit thereof for rabies virus. The kit consists of three parts such as a total RNA extraction template, a RT module, and a PCR module, and includes a RNA extraction agent, a RNA rinse, a reverse transcription premix system, an exterior nested PCR premix system, an interior nested PCR premix system, as well as the control of the reverse transcription (RT), and the control of PCR. The present invention is characterized in that RT-PCR is utilized to design the specific simplified primer based on the features of pandemic virulentstrains of RV to establish the process used to treat the sample and optimize the test procedures, thereby being capable of accomplishing the test on total 7 gene types of RV. The present invention ischaracterized in high sensitivity, strong specificity, simple and fast analytical procedures, and reliable analytical results, etc., obtains the information on sequence which can be directly used foranalysis on derivative relationship of the pandemic virulent strain, and is suitable for both the pathogenic diagnosis and the epidemiological research on rabies.

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 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 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 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 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.