29 results about "Fluorescent pcr" patented technology

This invention discloses a primer and probe set for detecting goat type C Kubuvir based on a temperature-controlled isolated fluorescent PCR platform. It includes upstream and downstream primers for goat type C Kubuvir and a fluorescent probe. This invention also provides a kit for detecting goat type C Kubuvir based on a temperature-controlled isolated fluorescent PCR platform and its application. This kit, used in conjunction with a temperature-controlled isolated PCR instrument, offers rapid detection, high specificity and sensitivity, and is easy to carry and store. It enables rapid and efficient on-site detection of goat type C Kubuvir, solving the technical problems of low detection rate, inability to rapidly and accurately detect various goat type C Kubuvir variants, and inconvenient operation in existing technologies. It shows promising application prospects.

This invention provides a primer-probe combination, reagents, methods, and applications for detecting Brucella vaccine strain RB51, belonging to the field of pathogen detection technology. The primer-probe combination for detecting Brucella vaccine strain RB51 of this invention includes a forward primer RB51-F, a reverse primer RB51-R, and a probe RB51-P; the forward primer RB51-F is shown in SEQ ID NO:1, the reverse primer RB51-R is shown in SEQ ID NO:2, and the probe RB51-P is shown in SEQ ID NO:3. The fluorescent PCR and digital PCR detection methods established based on the primer-probe combination of this invention have high sensitivity, good specificity, fast detection speed, and high throughput, enabling rapid detection of Brucella vaccine strain RB51, making differential diagnosis more accurate, economical, efficient, and convenient.

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 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 application belongs to the field of molecular biology, and particularly relates to a primer combination for detecting lung cancer EGFR mutant genes based on multiplex PCR-time of flight mass spectrometry and an application and a detection method. The primer combination is at least one EGFR mutant gene site in G719X (2155G>T, 2156G>C, 2155G>A), S768I (2303G>T), 19del (2236_2250del15, 2240_2257del18, 2235_2249del15), L858R (2573T>G), L861Q (2582T>A), T790M (2369C>T), 20ins (2300_2308dup, 2319_2320insCAC, 2310_2311insGGT). The application detects EGFR gene mutations by combining multiplex PCR and time of flight mass spectrometry, and has the advantages of low cost and fast detection speed compared with sequencing, fluorescent PCR and other technical means. The application further relates to an EGFR gene mutation detection kit.

The present application relates to the field of biotechnology, and particularly relates to a method for evaluating DNA methylation transformation efficiency. The present application can simply, quickly, directly and comprehensively evaluate DNA methylation transformation efficiency of the sample to be tested by detecting the methylation and non-methylation ΔCt values of four genes GNAS, GPR1, PAX6 and Actin respectively through conventional fluorescent PCR primer design, and calculating (2 ΔCtMSP +2 ΔCtUSP ) / (2 ΔCtMSP +2 ΔCtUSP +1). The method provided by the present application can be used for evaluation and quality control before carrying out methylation experiment or constructing methylation sequencing library, and the sample with low transformation efficiency can be detected in time to avoid carrying out subsequent experiment and causing resource waste such as experimental cost.

The present application provides a method for quantitative detection of rice purity and a kit for quantitative detection of rice purity. The method for quantitative detection of rice purity provided by the present application comprises: 1) extracting rice sample DNA; 2) performing PCR amplification using primers that can amplify the sequence of the difference fragments of fragrant rice and non-fragrant rice; and 3) performing quantitative analysis to determine the purity of fragrant rice in the rice sample. The present application utilizes the specific sequence of fragrant rice that is different from non-fragrant rice, designs efficient and sensitive fragrant rice specific primers and probes, performs real-time fluorescent PCR amplification on sample DNA, and performs quantitative analysis on the amplification data by setting an endogenous reference gene and a reference sample, thereby quantitatively determining the purity of fragrant rice. The quantitative detection of rice purity based on molecular biology effectively solves the problem of quantitative identification of non-fragrant rice mixed in fragrant rice.

This invention belongs to the field of molecular diagnostic technology and discloses a premixed reagent solution suitable for multiplex fluorescent PCR systems, its preparation method, and its application. The premixed solution uses purified water as a solvent and 2 mol / L sorbitol, 25% (w / v) trehalose, 2% (w / v) glycine, 20% (w / v) dextran, and stock solution of Tween-20 as raw materials. The preparation method involves mixing the components thoroughly and then drying them in a hot air drying oven to obtain a transparent gel-like product. This product is compatible with multiplex fluorescent PCR using multiple primers and probes and multi-channel detection. It has advantages such as low hygroscopicity, good resolubility, and excellent stability. It maintains excellent amplification efficiency and detection sensitivity even after room temperature storage, requires no cold chain transportation, and is convenient to use. Furthermore, the process is simple, cost-effective, and has good batch reproducibility, making it widely applicable in nucleic acid amplification scenarios such as clinical diagnosis and food safety monitoring.

The application discloses a mycoplasma pneumoniae 23S rRNA gene A2063G drug resistance mutation detection method based on a CRISPR-LbCas12a system and belongs to the technical field of molecular diagnosis. The detection method can accurately distinguish single base differences such as A2063 and G2063, effectively avoids mismatch, and ensures the accuracy of results. Relying on a one-step reaction of RPA and CRISPR, the total time consumption is less than 45 minutes, and the waiting time is significantly shortened. The whole reaction is carried out under constant temperature conditions, does not need to rely on complex equipment such as a thermal cycler, is easy to operate, is very suitable for the application of primary medical institutions and instant test scenes. The cost control advantage is outstanding, the reagent cost is about 1 / 50 of second-generation sequencing and 1 / 5 of fluorescent PCR, and the economy is extremely good. In addition, the application technology shows good multiplex detection potential and wide application prospect.

The application discloses a method for identifying authenticity of Cordyceps sinensis and products thereof based on fluorescent duplex PCR, and relates to the technical field of molecular identification of traditional Chinese medicinal materials. The method comprises the following steps: extracting total nucleic acid from a sample to be tested; using a fungal specific primer pair and an insect specific primer pair in the same PCR reaction tube, performing duplex PCR amplification by means of SYBR Green dye method, and collecting a fluorescent signal, the fungal specific primer pair is targeted at an ITS sequence of Cordyceps sinensis, and the insect specific primer pair is targeted at a COI gene sequence of host Thaumastia bennetti larvae; and determining results according to amplification curves and melting curves. The application provides a primer combination which can realize balanced and specific synchronous amplification of two target points at a universal annealing temperature of 60 DEG C, and breaks through the bottleneck of compatibility of duplex fluorescent PCR amplification caused by the conflict between the high GC content of the fungal ITS region and the low GC content of the insect COI region. The method is closed tube operation throughout, does not need gel electrophoresis and fluorescent probes, and has the advantages of high accuracy, convenient operation and low cost, and can be widely applied to authenticity identification of Cordyceps sinensis and deep processing products thereof.

This invention provides a lyophilized PCR reagent and kit for detecting monkeypox virus nucleic acid. Specifically, through extensive research, this invention has obtained a highly sensitive PCR amplification reagent for detecting monkeypox virus nucleic acid. This PCR amplification reagent can maintain the performance stability of the PCR reagent before and after lyophilization, and is therefore suitable for preparing lyophilized PCR reagents. This solves the problems of cumbersome preparation process, large preparation error, poor reaction repeatability, and high cost of product storage and transportation for fluorescent PCR reaction reagents.

The application provides a multiplex real-time fluorescent PCR reaction system based on probe differentiation design, corresponding fluorescent probes are designed based on target genes, the fluorescent probes are labeled with 1-3 fluorescent groups and 1-3 quenching groups, and the number and / or labeling position of the fluorescent groups and / or quenching groups carried by the fluorescent probes of different target genes are different. The reaction system can further carry multi-dimensional data analysis and a neural network model based on experience learning, unknown sample target gene sample types can be quickly and accurately judged, and accurate differentiation of multiple target genes in a single fluorescent channel can be realized.

The application discloses a real-time fluorescent PCR detection primer pair and probe combination of Nocardioides nucleic acid, a kit, a method and application, a nucleotide sequence of an upstream primer F of the primer pair is shown as SEQ ID:NO.1, and a nucleotide sequence of a downstream primer R is shown as SEQ ID:NO.2; the probe is a TaqMan probe with a fluorescent group FAM at a 5' end and a fluorescent quenching group BHQ1 at a 3' end, and a nucleotide sequence of the probe is shown as SEQ ID:NO.3. 0 The application can stably detect the Nocardioides at a low detection concentration of 5.62x10 0 copy / μL, can quickly, accurately and quantitatively detect the Nocardioides, has good specificity and can detect different sub-species of the Nocardioides, and has good universality.

This invention proposes a single-tube detection reagent for eight target nucleic acids, primer and probe compositions, and their applications, belonging to the field of genetic engineering technology. The eight-target nucleic acid detection kit (fluorescent PCR method) for influenza A virus, influenza B virus, novel coronavirus N gene, novel coronavirus O gene, norovirus GI type, norovirus GII type, respiratory syncytial virus, and capsicum mottle virus described in this invention can simultaneously detect eight pathogen targets: influenza A virus, influenza B virus, novel coronavirus N gene, novel coronavirus O gene, norovirus GI type, norovirus GII type, respiratory syncytial virus, and capsicum mottle virus. Parallel detection of eight pathogen targets can be achieved through a single-tube reaction, reducing the time wasted due to incorrect reagent selection caused by target misidentification, and reducing the consumption of consumables and instrument space required by existing multi-tube reactions. This provides efficient detection technology support for the rapid identification of pathogens in wastewater or the environment.

The present application relates to the technical field of plant molecular breeding, and particularly relates to a KASP molecular marker related to soybean alkali tolerance and application thereof. The KASP molecular marker corresponds to a Chr20_25666202 site in a soybean genome, the Chr20_25666202 site is located in a gene Glyma.20G072500 interior, and a C to T nucleotide mutation exists; the alkali tolerance of a soybean with a CC genotype of the Chr20_25666202 site is significantly higher than that of a soybean with a TT genotype, and the application of the KASP molecular marker in soybean alkali tolerance identification or molecular marker assisted breeding. Advantages lie in: solving the pain points of phenotype identification being easily interfered by environment, existing genetic markers being scarce, and breeding selection efficiency being low in alkali tolerance breeding; through an allele specific fluorescent PCR technology, rapid and accurate determination of alkali tolerance genotype is realized, and individual screening of alkali tolerance can be completed at the seedling stage without long-term field stress identification.

This invention relates to the field of biotechnology and discloses a multiplex fluorescent PCR detection system and its applications. The multiplex fluorescent PCR detection system includes 22 primer pairs, and discloses primer sequences targeting 22 sites, used to simultaneously amplify 15 detection sites and 7 internal reference sites. The multiplex fluorescent PCR detection system provided by this invention achieves effective detection of multiple methylation sites by combining capillary electrophoresis detection. Combined with corresponding detection methods and result interpretation methods, it enables highly sensitive and specific detection of colorectal cancer, possessing significant clinical application value and broad market prospects.

The application relates to the technical field of molecular biology detection, and discloses a z. laticeps environmental DNA detection kit, which comprises a primer probe composition for real-time fluorescent PCR detection designed based on a z. laticeps mitochondrial Cytb gene, the composition comprises an upstream primer, a downstream primer and a TaqMan-MGB probe, the nucleotide sequence of the upstream primer is shown in SEQ ID No. 1, the nucleotide sequence of the downstream primer is shown in SEQ ID No. 2, and the nucleotide sequence of the TaqMan-MGB probe is shown in SEQ ID No. 3; the 5' end of the TaqMan-MGB probe is modified with a fluorescent reporter group, and the 3' end is coupled with a small groove binder and a non-fluorescent quenching group; the application creates a special primer probe composition and a complete detection system for z. laticeps environmental DNA detection, realizes high specificity and high sensitivity detection of z. laticeps, and uses z. laticeps specific mitochondrial genes as detection targets; the TaqMan-MGB probe can accurately identify slight differences in gene sequences and effectively distinguish z. laticeps from closely related species in the same region.

This invention discloses a TaqMan real-time fluorescent PCR detection primer and probe set, kit, and detection method for Cuscuta pentaphyllum. The primer and probe set includes: an upstream primer C. pan F, whose nucleotide sequence is shown in SEQ ID NO.1; a downstream primer C. pan R, whose nucleotide sequence is shown in SEQ ID NO.2; and a TaqMan probe C. pan P, whose nucleotide sequence is shown in SEQ ID NO.3. The primers and probe target specific fragments of the rbcL gene sequence of Cuscuta pentaphyllum, achieving specific recognition of Cuscuta pentaphyllum through sequence complementarity matching. This avoids false positive results caused by cross-reactions with closely related species and host plants at the molecular targeting level, effectively solving the problem of difficulty in distinguishing closely related species in traditional morphological identification. Compared with traditional morphological identification and conventional PCR, this invention also features high detection efficiency, short processing time, simple operation, and wide applicability. It is suitable for precise identification in professional laboratories and can also meet the practical applications in grassroots quarantine and field monitoring scenarios.

The application discloses a multiplex fluorescence PCR kit and a detection method for multiple pathogenic microorganisms and drug-resistant genes. The kit comprises primers and fluorescence probes for detecting target pathogenic microorganisms and drug-resistant genes, a PCR reaction premix, positive quality control samples and negative quality control samples. Multiple fluorescence channels are used to mark corresponding detection targets, and the amplification of different targets is monitored by monitoring the change of fluorescence signals in different fluorescence channels, so that the detection of 96 pathogenic microorganisms and drug-resistant genes is realized. The multiplex fluorescence PCR kit can simultaneously detect multiple pathogenic microorganisms and drug-resistant genes, and has high accuracy, strong specificity, high sensitivity, rapidness and reliable results.

This invention relates to the field of detection kit technology, specifically to an African swine fever virus pathogen detection kit and detection method. This invention provides an African swine fever virus detection kit and detection method integrating specific primers and probes, a multiplex quality control system, and standardized operating procedures. By optimizing the nucleic acid extraction process, establishing a dual-channel fluorescent PCR detection system, and defining clear judgment criteria, it achieves high sensitivity and high specificity detection of African swine fever virus nucleic acid and full-process quality monitoring. It can not only accurately identify virus-positive samples but also effectively exclude false negative results through an internal standard system. This solves the key problems of insufficient specificity and low reliability of traditional detection methods, providing a stable and reliable complete technical solution for the clinical diagnosis and prevention and control of African swine fever.

The application discloses a probe primer group, a kit and a detection method for detecting European dodder, wherein the probe primer group comprises an upstream primer Ce-rbcL-F, a downstream primer Ce-rbcL-R and a TaqMan probe Ce-rbcL-P; the nucleotide sequence of the upstream primer Ce-rbcL-F is shown as SEQ ID NO. 1; the nucleotide sequence of the downstream primer Ce-rbcL-R is shown as SEQ ID NO. 2; and the nucleotide sequence of the TaqMan probe Ce-rbcL-P is shown as SEQ ID NO. 3. The primer probe group is designed based on a specific region of the rbcL gene of European dodder, and specific identification of European dodder can be realized by combining an optimized real-time fluorescent PCR reaction system and conditions, namely, the application has the characteristics of rapidness, strong specificity, high identification accuracy, effectively solves the problem that near relatives are difficult to distinguish in traditional morphological identification, and avoids false positive misjudgment. Compared with traditional morphological identification and conventional PCR, the application also has the characteristics of high detection efficiency, short time consumption, simple operation and wide applicability, and can be applied to not only professional laboratory accurate identification, but also practical application of scenarios such as primary quarantine and field monitoring.

This invention, entitled "A Primer-Probe Combination for Simultaneous Detection of AMB, VZV, and HSV-1, and its Detection Method, Application, and Product," belongs to the field of molecular biology detection technology. The technical problem it aims to solve is that existing ocular infection detection technologies cannot simultaneously detect AMB, VZV, and HSV-1 in a single tube, or only cover the virus and miss AMB, or require multiple tubes, have unclear identification, insufficient sensitivity, and are difficult to adapt to small ocular samples, failing to meet the needs of rapid and accurate clinical diagnosis. The key technical solution is to provide specific primers and probes targeting the 18S rRNA gene of AMB, the ORF62 gene of VZV, and the UL30 gene of HSV-1, combined with internal quality control primers and probes, using four different fluorescent labels to establish a single-tube multiplex real-time fluorescent PCR detection method, and preparing a matching kit to achieve simultaneous detection and identification of the three pathogens. This invention features high sensitivity, specificity, and strong anti-interference ability.

This invention discloses a rapid PCR detection kit and method based on a Staphylococcus aureus-specific protein target gene, belonging to the field of microbial molecular biology detection technology. The kit includes primer pairs and fluorescent probes for amplifying the Staphylococcus aureus-specific protein target gene. The kit also includes PCR reaction buffer, DNA polymerase, nuclease-free water, positive control, and negative control. This rapid PCR detection kit and method based on a Staphylococcus aureus-specific protein target gene, using a primer-probe combination with specific protein 2 reagent, validated 216 clinical isolates of Staphylococcus aureus confirmed by national standards, achieving a 100% positive rate. In contrast, the positive rate of the food industry standard fluorescent PCR method is only 87.0%, and the cosmetic industry standard fluorescent PCR method is 98.1%, thus solving the problem of missed detection in existing technologies.

Provided are primers and probes for single-tube multiplex fluorescent PCR detection of Treponema pallidum, herpes simplex virus 1 and herpes simplex virus 2, a fluorescent PCR kit, and the use thereof. The primers comprise specific sequences for Treponema pallidum, herpes simplex virus 1 and herpes simplex virus 2, respectively, which sequences have no cross-reaction. The specific sequences of the primers and probes are as shown in SEQ ID NO:1 to SEQ ID NO:9. A plurality of sexually transmitted pathogens such as Treponema pallidum, herpes simplex virus 1, and herpes simplex virus 2 can be detected simultaneously.

This invention discloses a triple lyophilized fluorescent PCR rapid detection kit for canine distemper virus, canine coronavirus, and canine parvovirus, belonging to the field of rapid detection kit technology. It includes a housing, protective components, and a sample collection mechanism. The back of the housing is symmetrically fitted with hinges via bolts. This triple lyophilized fluorescent PCR rapid detection kit for canine distemper virus, canine coronavirus, and canine parvovirus, through its primer, fluorescent probe, and reagent carrier components and sample collection mechanism, enables the rapid screening of three highly pathogenic canine viruses (canine distemper virus CDV, canine coronavirus CCV, and canine parvovirus CPV) using a lyophilization process based on quantitative real-time PCR technology.

This invention discloses a vancomycin resistance gene multiplex detection system based on real-time fluorescence PCR, aiming to establish a TaqMan probe-based multiplex real-time fluorescence PCR detection method for evaluating five common vancomycin resistance gene subtypes in clinical isolates. vanA、vanB、vanC、vanD and vanM The presence of [a substance] is used to clarify the drug resistance mechanism of the strain and guide precise clinical medication. The detection method established in this invention has superior performance, wherein... vanA and vanM The limit of detection is 10 2 copies / µL, vanB、vanC and vanD 10 3 Copies / µL. No specific amplification was observed in non-target strains, vancomycin-sensitive Enterococcus faecalis, and template-free controls. The coefficients of variation of Ct values ​​for each target strain were <1.0% both intra- and inter-batch. With NGS results as a reference, the overall concordance of this system among 108 clinical isolates was 100.00% (108 / 108).