221 results about "TaqMan" patented technology

The invention belongs to the technical field of aquatic pathogen diagnosis, and particularly relates to a Taqman multiple fluorescent quantitative PCR (Polymerase Chain Reaction) detection system and kit for common vibrio pathogens in aquaculture as well as application and a detection method. The system comprises primers and probes for detecting four common vibrio pathogens in aquaculture, the 5'end of each probe is modified with different fluorophores, the 3 'end of each probe is modified with corresponding quenching groups, and the common vibrio pathogens comprise vibrio anguillarum Va, vibrio alginolyticus Val, vibrio harveyi Vh and vibrio scophthalmi Vsc.

The embodiment of the invention discloses a probe primer combination for identifying TaqMan real-time fluorescent quantitative PCR of ampullaria gigas, a kit and application. Comprising a probe, an upstream primer and a downstream primer, the nucleotide sequence of the probe is 5 '-FAM-CCTTCTCTATTACTACTATTA-MGB-3', the nucleotide sequence of the upstream primer is 5 '-CTTTCCGCGTCTAATAACATG-3', and the nucleotide sequence of the downstream primer is 5 '-CATCAGTTCCAGCACCACTC-3'. The TaqMan probe real-time fluorescent PCR probe and primer combination established by the invention has excellent specificity and sensitivity in the aspect of identifying the species of the ampullaria gigas, the detection accuracy is greatly improved, the detection time is shortened, the effects of stability, reliability, short period and low cost are achieved, and the primer combination has good application prospects in rapid and accurate identification of the ampullaria gigas. The method is of great significance in promoting related prevention and research work.

The invention designs a carbapenem drug-resistant gene nucleic acid fluorescent PCR method detection kit and application thereof, the kit uses Taqman fluorescent probe multiple amplification technology to detect carbapenem drug-resistant genes, the kit comprises a nucleic acid reaction liquid Mix1 and a nucleic acid reaction liquid Mix2, the nucleic acid reaction liquid Mix1 is used for detecting drug-resistant genes IMP, NDM and OXA48, the nucleic acid reaction liquid Mix2 is used for detecting drug-resistant genes IMP, NDM and OXA48, and the nucleic acid reaction liquid Mix2 is used for detecting drug-resistant genes IMP, NDM and OXA48. The nucleic acid reaction liquid Mix2 is used for detecting drug resistance genes VIM, KPC and OXA23. The method is high in detection specificity, and the detection result is visual and easy to interpret. Clinical doctors are assisted to judge respiratory tract pathogen infection more comprehensively and more accurately. The kit covers six carbapenem drug resistance genes, is more comprehensive and accurate in detection, and is high in specificity and sensitivity. Meanwhile, multiple sample types can be detected, a sputum sample, an excrement sample and a rectum swab sample are detected through paramagnetic particle extraction, and pure bacterial colonies are directly detected without extraction.

The invention discloses a nucleic acid combination product for detecting various respiratory pathogens, a kit containing the nucleic acid combination product and a method. The nucleic acid combination product comprises a primer pair and a probe, wherein the nucleotide sequences of the primer pair and the probe are respectively shown as SEQ ID NO: 1-21. According to the nucleic acid combination product, on the technical basis of Taqman fluorescent quantification, seven targets can be detected by one tube, so that the time and energy of operators are greatly shortened, and the nucleic acid combination product is convenient and rapid, is not easy to make mistakes, and is simpler and easier to understand in result interpretation.

The invention discloses a TaqMan real-time fluorescent quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection fluorescent probe and kit for corn necrosis stripe virus and application, and relates to the technical field of virus detection. The nucleotide sequence of the fluorescent probe provided by the invention is 5 '-FAM-CGCTGGTACATAGGATGGCAAC-TAMRA-3', and the nucleotide sequence of the fluorescent probe provided by the invention is shown in the description. The nucleotide sequence of the primer is as follows: an upstream primer: 5 '-CCTGCTCCTCTGCTATTCG-3'; a downstream primer: 5 '- The downstream primer is 5 '-CAGACTTCCCTTGGCAGTGT-3', and the downstream primer is 5 '- The kit at least comprises the fluorescent probe and the primer. The TaqMan real-time fluorescent quantitative RT-PCR detection method established by the invention is simple and convenient to operate, strong in specificity, high in sensitivity and good in repeatability, is not only suitable for qualitative identification, but also can realize accurate quantitative analysis; the sensitivity is at least 100 times higher than that of a common RT-PCR detection method. The problem that in the prior art, a TaqMan real-time fluorescent quantitative RT-PCR detection method for detecting the corn necrotic stripe virus is lacked can be solved.

The invention discloses a Taq DNA polymerase mutant with improved excision activity and application thereof, and belongs to the technical field of enzyme engineering. The invention aims to solve the technical problems of low probe cutting efficiency, weak fluorescence signal, limited detection sensitivity (especially for low-abundance targets), low reaction speed (high Ct value) and reduced specificity caused by insufficient 5 '-> 3' excision enzyme activity of wild Taq DNA polymerase in TaqMan probe method qPCR (quantitative polymerase chain reaction). According to the technical scheme, the Taq DNA polymerase mutant with the high 5 '-3' exonuclease activity is provided, the amino acid sequence of the Taq DNA polymerase mutant is shown as SEQ ID NO.2, the exonuclease activity of the mutant is improved by 77%, the cutting speed of a TaqMan probe is greatly increased, accumulation of fluorescence signals is accelerated, the Ct value is reduced, and the detection time is remarkably shortened.

Disclosed are a probe method-based kit and method for tracking a behavior trajectory of Acipenser dabryanus Dumeril released upstream of the Yangtze River. By analyzing mitochondrial DNA sequences of Acipenser dabryanus Dumeril and common fish species in the Yangtze River basin, a pair of specific primers ND5-F4 / ND5-R4 capable of specifically amplifying mitochondrial DNAs of Acipenser dabryanus Dumeril and a Taqman fluorescent probe ND5 Probe 4 are designed and screened. A kit composed of the specific primers, the Taqman fluorescent probe, and a ddPCR master mix is used to carry out ddPCR testing on environmental DNA samples collected before and after release, so that a behavior trajectory of the released Acipenser dabryanus Dumeril can be tracked and monitored, achieving "non-contact" tracking of the behavior trajectory of the released Acipenser dabryanus Dumeril, avoiding damage to fish bodies in the tracking process, leading to reduced tracking costs and improved accuracy of overall tracking.

The invention discloses a primer and probe composition for detecting nocardia seriolae based on TaqMan qPCR (quantitative polymerase chain reaction) and application of the primer and probe composition. According to the invention, a qualitative and quantitative detection method for nocardia seriolae is established by utilizing the specific primers and probes, and the method is used for carrying out qualitative detection on water bodies and fish bodies of farms of snakeheads and hybrid snakeheads in different regions and detecting different tissues after the hybrid snakeheads counteract the nocardia seriolae, and has a very good detection effect. According to the invention, the purposes of nocardia seriolae monitoring, nocardia disease diagnosis, pathological degree measurement and the like can be realized, a reliable detection tool can be provided for nocardia seriolae epidemiological investigation, and nocardia disease can be diagnosed in an early stage so as to guide culture management and reasonable medication treatment; and a research basis and a technical support are provided for exploring epidemic characteristics and pathological mechanisms of nocardia seriolae.

The embodiment of the invention discloses a fluorescent primer combination for identifying TaqMan real-time fluorescent quantitative PCR of trachemys scripta elegans, a kit and application. Comprising a probe, an upstream primer and a downstream primer, the nucleotide sequence of the probe is 5 '-FAM-TTACTCCGACTATCCAG-MGB-3', the nucleotide sequence of the upstream primer is 5 '-TCCTAGGCCTTGCTGGAATG-3', and the nucleotide sequence of the downstream primer is 5 '-TGAAATAGAATTCTATGCG-3'. The TaqMan real-time fluorescent quantitative PCR probe primer combination developed by the invention has the characteristics of strong specificity and high sensitivity in the aspect of identifying trachemys scripta elegans species, and the detection accuracy and reliability are greatly improved; powerful technical support is provided for effectively monitoring distribution and diffusion of trachemys scripta elegans, preventing and controlling damage of trachemys scripta elegans to the ecological environment and guaranteeing public health safety.

The invention relates to a primer probe combination and a kit for detecting eDNA of Chinese sturgeons and application of the primer probe combination and the kit. Specific primers and TaqMan probes are designed for a D-loop region of Chinese sturgeon mitochondrial DNA, and efficient specific amplification and absolute quantification of Chinese sturgeon eDNA in an environmental sample are realized by optimizing a reaction system and amplification conditions. Compared with the prior art, the method is simple and convenient to operate, does not need to perform harmful sampling on Chinese sturgeon individuals, is suitable for resource monitoring and protection evaluation in a large-range and dynamic environment, and has high sensitivity, high accuracy and high practical value.

The invention relates to the technical field of typing identification, in particular to a primer and probe combination for typing identification of mycobacterium abscessus subspecies and a method and application of the primer and probe combination. The primer and probe combination comprises three subspecies specific amplification primer pairs and three subspecies specific TaqMan fluorescent probes; the sequences of the three pairs of subspecies specific amplification primers are SEQ ID NO. 1 to SEQ ID NO. 6; wherein the TaqMan fluorescent probe comprises three TaqMan fluorescent probes corresponding to mycobacterium abscessus subtypes, two ends of each TaqMan fluorescent probe are respectively provided with a fluorophore and a quenching group, and each sequence is SEQ ID NO.7 to SEQ ID NO.9. The invention further discloses a kit with the PCR reaction liquid, and by means of the PCR reaction liquid, the mycobacterium abscessus can be rapidly and accurately detected and identified, and subspecies typing identification can be conducted on the mycobacterium abscessus.

The invention provides a kit for detecting human BRAF gene V600E and TERT gene promoter mutation, and belongs to the technical field of biology. The kit comprises a primer, a probe, a 5 * PCR (Polymerase Chain Reaction)-Enhancer, a 10 * PCR Buffer, dNTP (Deoxyribonucleoside Triphosphate) Mix, MalTaq DNAPlyase, magnesium ions, DEPC (Diethylpyrocarbonate) water, a positive control and a negative control. According to the present invention, the ARMS-PCR detection technology is adopted to detect the specific gene mutation, and the target mutation site is identified by designing the specific primer and the probe, such that the detection specificity and the detection sensitivity are improved; by combining with a Taqman probe technology, the fluorescent signal change in the PCR amplification process can be monitored in real time, so that whether mutation of a BRAF V600E site, a TERT C228T site and a TERT C250T site exists or not is accurately judged; according to the method, pre-amplification of 15 cycles is adopted, then quantitative amplification of 30 cycles is adopted, and when the initial amount of the target DNA is very low, the copy number of the target DNA can be increased, so that the detection accuracy and sensitivity are further improved.

The invention discloses TaqMan real-time fluorescent quantitative PCR (polymerase chain reaction) primers, a probe, a detection method and a kit for detecting oviductus ovatus and application of the TaqMan real-time fluorescent quantitative PCR primers and the probe. The primer comprises an upstream primer HO-F and a downstream primer HO-R, and the nucleotide sequences of the upstream primer HO-F and the downstream primer HO-R are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2. The nucleotide sequence of the probe HO-P is shown as SEQ ID NO.3, the 5'end of the probe HO-P is marked with a fluorescence reporter group FAM, and the 3 'end of the probe HO-P is marked with a quenching group BHQ1. The TaqMan real-time fluorescent quantitative PCR method established by using the primer and the probe can specifically detect the ovulus ovatus of sheep, and has no cross reaction with other bacteria of ovulus ovatus and 47 common bacteria of cattle and sheep. The method or the kit not only can be applied to rapid identification of oviductus ovatus, but also can be applied to qualitative, relative quantitative and absolute quantitative detection of oviductus ovatus in cattle and sheep clinical samples, has the advantages of strong specificity, high sensitivity, good repeatability and the like, provides a reliable method for rapid detection of oviductus ovatus infection of cattle, sheep and sheep, and has a wide application prospect. Good popularization and application prospects are realized.

The application relates to a primer probe composition, a kit and a detection method for specifically detecting Blattella germanica. The specific primer and TaqMan probe are designed and screened based on the specific sequence of the mitochondrial COI gene of Blattella germanica. The detection method has extremely high specificity, has no cross reaction with close species such as Periplaneta americana and Blattella germanica, has high sensitivity, the minimum detection limit is 1 fg / muL, and has good repeatability, with the variation coefficients of groups and between groups being less than 3%.

The invention belongs to the technical field of biological detection, and particularly relates to a primer probe pair, a microfluidic chip and a kit for detecting pathogens of vector infectious diseases based on a TaqMan microfluidic chip technology as well as a preparation method and application of the primer probe pair. Based on a TaqMan microfluidic chip technology, the invention provides a detection method for simultaneously detecting 28 kinds of pathogens of the disease-borne infectious diseases, and the related pathogens of the disease-borne infectious diseases comprise viruses, bacteria and parasites, and are wide in range and large in difference. The detection method provided by the invention has a low detection limit (5-20 copies / [mu] L); when the method is applied to an actual sample, the sensitivity, specificity, accuracy and precision are high, and compared with a gold standard method, the consistency is good; and compared with a traditional qPCR method, the method has the advantages that the detection time efficiency and the manpower and material consumption are greatly reduced, and the method is particularly suitable for disease monitoring and emergency disposal scenes with undefined pathogen spectrum, need of rapid and comprehensive troubleshooting and the like, and has a good application prospect.

The invention relates to a quadruple TaqMan real-time fluorescent quantitative PCR (Polymerase Chain Reaction) primer group and a probe group for simultaneously detecting cGPV (Complementary Glutathione Virus), MDPV (Minimal Disease Papilloma Virus), MDGPV (Minimal Disease Papilloma Virus) and SBDSV (Sequence Broadcast DSV) and a kit thereof. The sequences of the primer group and the probe group are respectively shown as SEQ ID NO.1-12. The research successfully develops and verifies a multiple TaqMan-MGB real-time fluorescent PCR method capable of simultaneously detecting and distinguishing four important waterfowl parvoviruses (cGPV, MDPV, MDGPV and SBDSV). The method has good specificity, repeatability and high sensitivity. Compared with a conventional PCR method, the multiple detection method has the advantages that the clinical waterfowl parvovirus detection rate is remarkably improved, mixed infection can be effectively recognized, and the clinical diagnosis efficiency is greatly improved.

The invention discloses an aptamer-based oxytetracycline fluorescent quantitative PCR (Polymerase Chain Reaction) detection kit as well as a preparation method and a detection method thereof, and relates to the technical field of analysis and detection.The detection method comprises the following steps: mixing and incubating a sample to be detected, an oxytetracycline specific aptamer, a complementary chain and a magnetic bead curing capture probe; magnetically separating out the mixed and incubated magnetic beads, curing and capturing the probe, and washing to obtain a magnetic compound; adding PCR reaction liquid containing the primer pair and a TaqMan probe into the magnetic compound, carrying out fluorescent quantitative PCR amplification, and monitoring a fluorescent signal in real time to obtain a Ct value; establishing a standard curve between the oxytetracycline concentration and the Ct value of the fluorescent quantitative PCR, and determining the oxytetracycline concentration in the sample to be detected according to the standard curve. Based on an indirect competitive mechanism, through signal conversion design, combination of oxytetracycline and the aptamer is converted into quantitative analysis of free sDNA, and through integration of three technologies of aptamer free recognition, magnetic bead targeted capture and qPCR signal amplification, the sensitivity and anti-interference capability of oxytetracycline detection are improved.

The invention discloses a primer, a TaqMan probe and a kit for carrying out fluorescent PCR (Polymerase Chain Reaction) detection on bovine-derived ureaplasma, and belongs to veterinary detection of animal epidemic diseases in the technical field of biological detection. The invention discloses a primer and a TaqMan probe which are used for carrying out fluorescent PCR (Polymerase Chain Reaction) detection on bovine-derived ureaplasma, and the primer and the TaqMan probe are an upstream primer U.diversum-F3, a downstream primer U.diversum-R3 and a TaqMan probe U.diversum-P3. The kit is simple and convenient in detection operation, strong in specificity, high in sensitivity and good in repeatability, and can be used for qualitatively detecting the bovine-derived ureaplasma; technical support is provided for detection of bovine-derived ureaplasma, epidemic situation epidemiological investigation, epidemic disease monitoring and purification, strain screening and quality monitoring and stable production and supply conservation in the vaccine production process, and the method plays an important role.

The invention belongs to the technical field of biology, and discloses a TaqMan primer probe combination for detecting virulent and attenuated Newcastle disease, which comprises a virulent primer RT, a virulent probe RT-probe, a universal primer SRT and a universal probe SRT-probe, the virulent primer RT comprises an upstream primer RT-F and a downstream primer RT-R, and the universal primer SRT comprises an upstream primer SRT-F and a downstream primer SRT-R; a TaqMan probe fluorescent quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection method for virulent and attenuated NDV (Newcastle Disease Virus) established by using the TaqMan primer probe combination has no cross reaction on amplification of common avian viruses AIV, FADV, IBV and IBDV and has good specificity; the lowest detection concentration of virulent and attenuated Newcastle disease virus is 101 copies / uL, which is 100 times higher than that of a conventional RT-PCR method, and the kit has good sensitivity; and compared with the conventional RT-PCR method, the detection rate is 100% and is superior to that of the conventional RT-PCR method, and the detection result is consistent with the sequencing result.

This invention relates to a method and its application for determining the plasmid content in a quadrivalent avian influenza DNA vaccine. Belonging to the field of molecular biology, this invention aims to provide a TaqMan quantitative real-time PCR method capable of quantitatively determining the content of each plasmid component in a quadrivalent DNA vaccine. Specifically, this invention provides a primer and probe composition for detecting the content of individual plasmids in a quadrivalent avian influenza (H5+H7) DNA vaccine based on TaqMan quantitative real-time PCR. The composition includes primers with nucleotide sequences shown in SEQ ID NO: 5-6, 8-9, 11-12, and 14-15, and TaqMan probes with nucleotide sequences shown in SEQ ID NO: 7, 10, 13, and 16. This primer and probe composition can quantitatively determine the content of each plasmid component in the quadrivalent DNA vaccine, exhibiting good specificity and reproducibility.

The invention discloses a virulent virus quadruple Taqman probe fluorescent quantitative PCR (Polymerase Chain Reaction) detection kit and application thereof, specific primers and corresponding fluorescent probes of four virulent viruses including Nipah virus, Hendea virus, Rift Valley fever virus and Congo fever virus are designed, and then conserved gene sequences of various viruses are selected by utilizing known virus genome sequences; the method comprises the following steps: constructing a standard recombinant plasmid containing a conserved gene sequence by using a gene cloning technology, then identifying the recombinant plasmid to prepare a plasmid standard substance so as to obtain a standard curve, evaluating the sensitivity, specificity and repeatability of a PCR (Polymerase Chain Reaction) method, and finally forming the detection kit by using a fluorescent quantitative PCR reaction system. The kit is simple and efficient in detection operation, can achieve the purpose of simultaneously detecting four virulent viruses in one PCR reaction tube, has the characteristics of high sensitivity, strong specificity and good repeatability, and can be widely applied to clinical sample detection.

The invention discloses a KIM-1 detection kit, which is designed based on an RT-LAMP method of Taqman, and comprises: a specific primer group comprising an outer primer and an inner primer; a TaqMan probe; the reaction enzyme mixture comprises BstDNA polymerase with strand displacement activity and AMV reverse transcriptase; the reaction buffer solution is prepared from Tris. HCl, KCl, (NH4) 2SO4 and Tween20; the quality control product comprises a positive control group, a negative control group and a blank plasmid control group. Compared with the prior art, the kit has the advantages that the KIM-1 detection kit is designed based on the RT-LAMP technology of Taqman, and quantitative detection of KIM-1 mRNA is achieved through the integrated reaction of reverse transcription, amplification and real-time fluorescence detection.

The application provides a digital PCR kit for detecting highland invasive species red-eared slider and bullfrog, a set of red-eared slider specific primer probes, which are composed of a primer pair for a specific segment of a red-eared slider mitochondrial CYTB gene and a red-eared slider specific TaqMan probe matched therewith; a set of bullfrog specific primer probes, which are composed of a primer pair for a specific segment of a bullfrog mitochondrial 12S rRNA gene and a bullfrog specific TaqMan probe matched therewith, and a set of internal standard (IPC) primer pairs and internal standard TaqMan probes for monitoring the nucleic acid extraction and amplification reaction process monitoring. The application is provided with the primer pair for the specific segment of the red-eared slider mitochondrial CYTB gene and the red-eared slider specific TaqMan probe, the primer pair for the specific segment of the bullfrog mitochondrial 12S rRNA gene and the bullfrog specific TaqMan probe, and a common internal standard to construct a triple detection reaction system, thereby avoiding the quantitative uncertainty caused by the single species detection in the prior art which needs to be carried out in multiple reactions or relies on a standard curve for relative quantification.

The embodiment of the invention discloses a TaqMan real-time fluorescent quantitative PCR probe primer combination for identifying tilapia mossambica, a kit and application. Comprising a probe, an upstream primer and a downstream primer, the nucleotide sequence of the probe is 5 '-FAM-CGCTATACAGGG-MGB-3', the nucleotide sequence of the upstream primer is 5 '-GACTTTCCCCCATATTC-3', and the nucleotide sequence of the downstream primer is 5 '-GGGGTAGACAGTTCACCCTGTA-3'. Based on difference of mitochondrial control areas of different fishes, the TaqMan probe real-time fluorescent PCR detection method established by designing specific oligonucleotide primers and probes can effectively distinguish tilapia homosambica from other fishes, has excellent specificity and sensitivity in the aspect of identifying tilapia homosambica species, and can be used for detecting the species of tilapia homosambica. The detection accuracy is greatly improved, meanwhile, the detection time is shortened, the effects of stability, reliability, short period and low cost are achieved, and the actual application requirements are met.

The application discloses a specific SNP site primer combination for identifying Taihe black-bone chicken varieties and application thereof. A chicken pan-genome containing a chicken reference genome GRCg7b and 2.49 Gb pan-sequence is taken as reference, WGS data of Taihe black-bone chicken and related chicken varieties is analyzed and screened to obtain two specific SNP sites, namely novel_th1 (G / T, dominant allele G) and novel_th2 (C / T, dominant allele C), wherein the T allele is medium to high frequency in most non-Taihe black-bone chicken varieties / strains. A FAM / VIC double-labeled TaqMan probe and a matching primer are designed based on the two sites to construct a real-time fluorescent quantitative PCR detection kit for joint typing of the two sites, and an amplification system and a Ct value judgment standard are established, so that 96 varieties / strains such as Taihe black-bone chicken, Zhushi chicken, other black-bone chicken, local chicken, white-feathered broiler and egg chicken can be quickly and accurately identified. The method has the advantages of high specificity, high sensitivity, simple operation and low cost, and is suitable for identification of authenticity of Taihe black-bone chicken and products thereof, purity monitoring and molecular-assisted breeding.

The invention relates to the technical field of molecular biology, and particularly discloses a porcine bocavirus G3 gene group SYBR Green I fluorescent quantitative PCR (polymerase chain reaction) detection primer and application thereof. A synthetic primer is designed according to a highly homologous conserved region of a VP1 gene of a PBoV-G3 gene group, an SYBR Green I fluorescent quantitative PCR method for detecting PBoV-G3 is established, and the SYBR Green I fluorescent quantitative PCR method has the advantages of strong specificity, high sensitivity and good repeatability. Compared with a TaqMan probe method, the method has the advantages that no complex and expensive probe is used, and the detection cost is saved. Compared with common PCR, SYBR Green I fluorescent quantitative PCR can directly quantify the virus load without treatment after amplification, so that the possibility of pollution is reduced, and the method is more suitable for clinical detection of the porcine bocavirus G3 gene group.

The invention relates to a primer group, a composition, a kit and a device for detecting pseudomonas aeruginosa. The composition comprises an upstream primer and a downstream primer of target double-stranded DNA, a TaqMan fluorescent probe, recombinase, T7RNA polymerase, transcriptase, ATP energy regeneration enzyme, melting enzyme, single-stranded binding protein and cutting enzyme. According to the invention, nucleic acid amplification enzyme and signal amplification enzyme are integrated together, the purpose of amplifying target double-stranded DNA to 109 times within 10-30 minutes under the condition of constant temperature of 25 DEG C is realized, and each nucleic acid amplification product can generate more than 10,000 fluorescence signals under the condition of constant temperature of 25 DEG C, so that efficient and rapid detection of samples is realized; according to the method, the omission ratio caused by false negative is extremely low, the operation is convenient, and the steps are simple.

The application provides a standard plasmid for detecting the copy number of exogenous genes of a recombinant CVA10 vaccine, and a preparation method and application thereof. The standard plasmid of the application comprises an endogenous gene MOX fragment SEQ ID No. 1, an exogenous gene P1 fragment SEQ ID No. 2 and an exogenous gene 3CD fragment SEQ ID No. 3 which are connected in series in a cloning vector. The application constructs the standard plasmid, adopts Taqman probe-fluorescence quantitative PCR method, and simply, quickly, absolutely quantitatively and accurately analyzes the copy number of inserted exogenous genes, so that the detection time of the determination of the copy number of exogenous genes of the recombinant CVA10 vaccine is effectively shortened, repeated operation is facilitated, and the determination of the copy number of exogenous genes of the recombinant CVA10 vaccine based on the Hansenula platform and the verification of the genetic stability of the exogenous genes of the recombinant CVA10 vaccine have a wide application prospect.

The invention discloses a dual real-time fluorescent quantitative TaqMan primer probe group, a kit and a detection method for simultaneously detecting sweet potato leaf curl virus and sweet potato arbuscular phytoplasma, aiming at nucleotide sequences of SPLCV and SPWB genome sequencing, corresponding specific TaqMan detection primer groups and probe primers are designed in conservative segments, and are used for specific detection of SPLCV and SPWB; the invention also designs a fluorescent quantitative kit based on the primer probe group. According to the primer and the probe group, the targets of SPLCV and SPWB can be detected at the same time through one-time reaction, rapidness and convenience are achieved, the detection efficiency is high, and meanwhile the high detection sensitivity of 3.3 copies / uL is achieved and is 100 times that of common PCR. The TaqMan QPCR detection technology established aiming at the SPLCV and the SPWB solves the problems that the existing SPLCV and SPWB detection method is time-consuming and labor-consuming, poor in specificity and sensitivity, easy to cause pollution and the like, and provides a technical basis for early diagnosis of the SPLCV and the SPWB.

The invention discloses a primer, a probe, a method and a kit for detecting CG methylation of a CpG island of an MGMT gene. The primer comprises a specific primer for amplifying DNA fragments of five CG sites on the CpG island of the MGMT gene from sample nucleic acid and a fluorescent probe for presenting the obtained nucleic acid fragments. According to the primer, the probe, the method and the kit, the Taqman probe technology is adopted, the methylation frequency of the MGMT gene related to treatment medication can be detected, so that a basis is provided for clinical medication, and the method is good in specificity, high in accuracy and high in flux.