122 results about "TaqMan" patented technology

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 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.

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 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 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 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 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 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 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 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.

The application provides a primer probe composition for detecting genetic stability of cells and application thereof, the primer probe composition comprises primer pairs SEQ ID NO:1-2 and a probe SEQ ID NO:3 of a VEGF light chain gene, and further comprises primer pairs SEQ ID NO:4-5 and a probe SEQ ID NO:6 of a VEGF heavy chain gene. The application quantitatively detects the antibody light and heavy chain gene copy numbers in cells of different culture generations by fluorescence quantitative PCR based on a Taqman probe method, extracts cell genomic DNA and performs PCR amplification, and prepares a target gene standard product from the amplification product, thereby significantly improving the specificity and accuracy of quantitative detection of the gene copy number.

The application relates to the technical field of animal virus molecular detection, and particularly discloses a multiple detection method for ASFV, PCV2 and PCV3 suitable for rapid real-time fluorescent PCR. Specific primers and TaqMan probes are designed according to the B646L gene of ASFV and the ORF1 gene conservative regions of PCV2 and PCV3, and the sequences are shown as SEQ ID NO:1-9. The primers and probes are optimized: avoiding strong GC anchoring at the 3' end of the primers, continuous AT series and G base at the 3' end of the probes, and shortening the length of the probes to adapt to rapid real-time fluorescent PCR. The minimum detection limit of the three viruses is not higher than 5 copies per muL, the detection is completed within 30 minutes, there is no cross reaction with 11 common pig pathogenic agents, the coefficient of variation is less than 2%, the sensitivity is high, the specificity is strong, the method is rapid and convenient, and is suitable for rapid diagnosis of mixed infection of ASFV, PCV2 and PCV3.

This invention provides a microfluidic chip for the multiplex differentiation and diagnosis of different avian influenza virus subtypes and its application. Specifically, it is a microfluidic chip based on TaqMan probes that can simultaneously and rapidly differentiate and diagnose 25 avian influenza subtypes, enabling high-throughput simultaneous detection of multiple gene targets. This invention utilizes high-throughput microfluidic chip technology to simplify the differential diagnosis procedure for avian influenza virus subtypes, resulting in more accurate and time-saving results. It is more suitable for rapid clinical diagnosis of avian influenza, providing technical support for rapid clinical detection, diagnosis, and prevention of avian influenza, and is of great significance for epidemic prevention and control and safeguarding public health security.

The invention discloses a TaqMan probe qPCR (quantitative polymerase chain reaction) detection primer group for detecting pathogenic vibrio alginolyticus, a kit and application, and belongs to the technical field of molecular biology and pathogenic microorganism detection. The method comprises the following steps: designing a pair of specific primers and probes by taking the vops gene of the vibrio alginolyticus as a new target, connecting the vops gene with plasmids by amplifying the vops gene, constructing a recombinant plasmid standard substance, carrying out qPCR detection on the copy number of the vops gene of the vibrio alginolyticus by utilizing the designed specific primers and probes, and establishing a corresponding relationship between the vibrio alginolyticus and the vops gene. And the vibrio alginolyticus can be quantified through the copy number of the vops gene. On the basis of novel molecular target detection, no amplification signal exists for a detection sample which does not contain vibrio alginolyticus, and the specificity is good; the method is rapid and accurate in detection and high in sensitivity, the lower detection limit is as low as 100-level copies, non-specific amplification can be avoided, and operation is simple and rapid.

This invention discloses a feline calicivirus nucleic acid detection kit and detection method. The detection kit includes: (1) a nucleic acid enrichment module and (2) a TaqMan fluorescent PCR detection module. The beneficial effects are that the sensitivity is significantly improved, the operation is simplified, the functions are integrated, the pathogenicity is classified in one step (distinguishing between highly pathogenic strains and classic strains), the stability is excellent, and the specificity is strong.

The invention relates to a multiplex fluorescent quantitative PCR (polymerase chain reaction) detection method for streptococcus equi subsp. Zooepidemicus virulent strains causing swine streptococcosis, and belongs to the technical field of bacterial detection. The invention provides a primer probe group which can be used for simultaneously detecting M protein variable region genes (szm-variant) of streptococcus equi subsp.zooepidemicus, conserved genes (comB) in subsp.zooepidemicus and central nervous system infection related virulence genes (bifA) of streptococcus equi subsp.zooepidemicus through Taqman probe triple fluorescent quantitative PCR (Polymerase Chain Reaction), and is shown as SEQ ID NO.1-9 in a sequence table. The multiplex fluorescent quantitative PCR detection method for the streptococcus equi subsp. Zooepidemicus virulent strain causing the swine streptococcosis can complete nucleic acid amplification and result judgment within 1 hour. According to the detection method, the amplification result is judged through the threshold cycle number (Ct value), electrophoresis detection is not needed, and the problems of pollution, false positive and the like of an amplification product can be avoided. The detection method provided by the invention has the characteristics of simplicity, convenience and rapidness in operation, high specificity, high sensitivity and good stability.

The application discloses a parrot-origin reovirus RT-qPCR specific detection primer and a detection method. The primer probe composition takes a S1 gene conservative region of the parrot-origin reovirus as an amplification target, and comprises a specific upstream primer, a downstream primer and a fluorescent probe. The nucleotide sequence of the upstream primer is shown as SEQ ID NO:1, the nucleotide sequence of the downstream primer PSRV-S1-R is shown as SEQ ID NO:2, and the nucleotide sequence of the fluorescent probe PSRV-S1-Probe is shown as SEQ ID NO:3. The application designs specific primers and a TaqMan probe for the parrot-origin reovirus, has an amplification signal for the PSRV, has no cross reaction with common pathogens such as APV, BFDV, FAdV, ARV, NDRV and parrot fever chlamydia, has good specificity, is accurate in quantification, has good repeatability and stability, and is simple in operation and high in flux.

The invention provides a real-time fluorescent quantitative PCR (Polymerase Chain Reaction) detection method for total RNA (Ribonucleic Acid) residual quantity of escherichia coli. A reaction system comprises 3-5 mu L of Taqman RT-PCR Buffer (5 *), 1-3 mu L of TaqMan Enzyme Mix (10 *), 0.3-0.5 mu L of ROX Reference Dye (50 *), 1-1.5 mu L of 8-12 mu M forward specific primer, 1-1.5 mu L of 8-12 mu M reverse specific primer, 0.5-0.8 mu L of 8-12 mu M specific probe, 4-6 mu L of standard substance or to-be-detected sample and RT-PCR-grade water which is supplemented to 20 mu L, and a reaction procedure is as follows: the temperature is 52-54 DEG C for 4-6 minutes; the temperature is 94-96 DEG C for 18-22 s; the detection time is 4-6 seconds at the temperature of 94-96 DEG C, 38-42 seconds at the temperature of 56-58 DEG C and 44-46 cycles, and the detection limit of the method is as low as 2 pg / mL.

The application provides a molecular marker which can be used for distinguishing and identifying Schistosoma japonicum and Trichobilharzia orientalis, and constructs a qPCR detection method (SYBR Green dye method and TaqMan probe method) which has high sensitivity, strong specificity and can distinguish and identify Schistosoma japonicum and Trichobilharzia orientalis, and can accurately and quickly distinguish Schistosoma japonicum and Trichobilharzia orientalis.

The invention discloses a pertussis Bordetella prn gene insertion deletion detection kit and a detection method, the kit comprises a reaction liquid, a positive reference substance A, a positive reference substance B, a positive reference substance C and a blank reference substance, the reaction liquid contains MgSO4, dNTP, DMSO and taq enzyme with specific concentrations, and specific primers and probes aiming at a ptxP gene, a prn gene and an IS481 insertion type prn gene; and the probes are respectively marked with different fluorescent dyes. The detection method is based on a TaqMan probe method fluorescent quantitative PCR (Polymerase Chain Reaction) technology, a single-tube triple amplification reaction is combined with a specific amplification system and reaction conditions, and the wild type, prn gene insertion type and deletion type bordetella pertussis can be quickly, specifically and sensitively identified. The method solves the problems that in the prior art, rapid typing cannot be achieved, operation is tedious, and consumed time is long, and is suitable for clinical rapid diagnosis and large-scale epidemiological monitoring.

The invention discloses a kit for detecting intestinal core bacteria by a TaqMan real-time fluorescent quantitative PCR (Polymerase Chain Reaction) method, and belongs to the technical field of kits. The kit disclosed by the invention comprises standard positive templates of nine common intestinal core bacteria and nine groups of specific primers and probes which are required in a PCR amplification process and correspond to the standard positive templates. The kit disclosed by the invention is simple, convenient and rapid to operate; the specificity is good and the sensitivity is high; complicated raw information data analysis does not need to be carried out; the intestinal core bacteria can be detected and quantified without high-throughput sequencing, and three bacteria can be detected in one reaction hole, so that the cost is greatly reduced. The kit provided by the invention can be used for rapidly and quantitatively detecting nine intestinal core bacteria including bifidobacterium, lactobacillus, prevotella, bacteroides and the like, can partially replace a high-throughput detection method which is higher in cost and more complicated to operate according to research and diagnosis purposes, and is suitable for being widely popularized in clinical detection and laboratory research.

The invention discloses a primer probe combination and a kit for detecting staphylococcus caprae and application of the primer probe combination and the kit, and belongs to the technical field of biology. Aiming at the defects of long time consumption and insufficient specificity of the existing staphylococcus caprae detection method, a specific primer and a TaqMan probe with a fluorescent label are designed based on a conserved region of an intercellular adhesion protein A gene (icaA) of the staphylococcus caprae, and the specific primer and the TaqMan probe have no cross reaction with other staphylococcus bacteria with similar genetic relationship, so that high-specificity detection can be realized. The matched kit comprises a primer probe combination, a PCR reaction component, a positive quality control product and a negative quality control product, the detection process is simple and convenient to operate, the sensitivity is high, an effective means is provided for rapid detection of staphylococcus caprae, and the kit can be applied to the fields of blood infection diagnosis, food pollution monitoring, clinical sample detection and the like.

The invention belongs to the technical field of virus detection, and particularly relates to a TaqMan real-time fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) primer probe group for detecting capsicum beet western yellowing virus and application of the TaqMan real-time fluorescent quantitative RT-PCR primer probe group. The TaqMan real-time fluorescent quantitative RT-PCR primer probe group comprises an upstream primer, a downstream primer and a probe; the sequence of the upstream primer is as shown in SEQ ID NO. 1; the sequence of the downstream primer is as shown in SEQ ID NO. 2; and the sequence of the probe is as shown in SEQ ID NO. 3. The TaqMan real-time fluorescent quantitative RT-PCR primer probe group is suitable for fluorescent quantitative RT-PCR amplification, has good specificity and high sensitivity, and is suitable for wide popularization and application in the field of plant disease diagnosis. The sensitivity of a real-time fluorescent RT-PCR detection method established based on the fluorescent probe or the kit is at least 100 times higher than that of a common RT-PCR method; and the detection method can specifically identify the pepper beet western yellowing virus.

The invention discloses a triple qPCR method and kit for simultaneously detecting EHP, VpTPD and VpAHPND of shrimps, and belongs to the technical field of pathogen detection. According to the invention, a qPCR primer and a TaqMan probe for detecting EHP, VpTPD and VpAHPND of shrimps are respectively designed, and the nucleotide sequences of the qPCR primer and the TaqMan probe are as shown in SEQ ID NO: 1-9. By utilizing the primer and the probe, qualitative and quantitative detection of three pathogens, namely EHP, VpTPD and VpAHPND, can be realized at the same time through a triple qPCR (quantitative polymerase chain reaction) method. The triple qPCR method for simultaneously detecting VpAHPND and VpTPD is established on the basis of local pathogen strains for the first time, has the characteristics of high efficiency and convenience, solves the problem of time and labor waste caused by multiple times of single fluorescent quantitative PCR, and effectively reduces the cost of manpower and material resources; meanwhile, the molecular crowding agent is added as a component for improving the amplification efficiency, so that the detection efficiency is improved, and the reliability of the detection limit is improved; the method also has good repeatability, sensitivity and specificity.

The invention discloses a primer probe combination for detecting parvovirus B19. According to the invention, a method for simultaneously detecting double targets of the NS1 gene and the VP1 gene of the B19 virus is established, the detection accuracy is improved, the clinical omission ratio is reduced, meanwhile, the result credibility is increased, and unnecessary confirmation experiments are reduced. Meanwhile, the method using the MGB-TaqMan fluorescent probe has the advantages of rapidness, sensitivity, easiness in operation and the like, the double-target synergism is good, and the clinical reinspection condition can be reduced.

This invention discloses primer-probe combinations, kits, and applications for the dual-system detection of TobRFV (Tobacco Brown Fruit Virus) using TaqMan and EmDEA, belonging to the field of viral molecular diagnostics technology. Based on highly conserved and specific viral core target sequences selected through screening, this invention constructs primer-probe combinations suitable for both TaqMan qPCR and EmDEA detection systems. These combinations target two different genes of ToBRFV (p126 and MP), not only specifically detecting ToBRFV and exhibiting no cross-reactivity with viruses of the same genus such as TMV, ToMV, and ToMMV, but also broadly covering the vast majority of ToBRFV isolates. This invention effectively reduces the risk of missed detection in single-target detection and has significant practical application value for accurate quantification of this virus in the laboratory and rapid field quarantine.