13 results about "Duplex pcr" patented technology

The invention belongs to the field of plant molecular genetics, and relates to a spirodela polymorphic microsatellite molecular marker primer and application thereof. The kit comprises 10 pairs of microsatellite primers which form 5 groups of duplex PCR systems, and the kit can be applied to population genetic diversity analysis of spirodela polyrhiza. The population genetic diversity analysis comprises the following steps: collecting a sample of spirodela polyrhiza to be identified; extracting spirodela polyrhiza genome DNA from the spirodela polyrhiza sample; taking the extracted DNA as a template, and carrying out PCR (Polymerase Chain Reaction) amplification on the DNA template by using the screened 10 pairs of microsatellite primers; carrying out electrophoresis detection on a PCR product, and then carrying out silver staining; the silver staining result is digitally analyzed, the UPGMA clustering analysis chart is drawn by genetic analysis software, the clustering analysis chart of the spirodela polyrhiza is identified according to the clustering analysis result, and the method has huge popularization and utilization value.

The invention belongs to the technical field of crop molecular markers, and particularly discloses a molecular marker for identifying the salt tolerance of brassica napus in the germination period and application of the molecular marker. The molecular marker is located at the position of an A02 chromosome scafoldA028707654 of a brassica napus reference genome ZS11.v10, and the allelotype of the molecular marker is wild type C or mutant type CATATT insertion. The invention also provides a specific primer group for detecting the molecular marker, and the salt tolerance (sensitive type or tolerance type) of the brassica napus line can be quickly identified according to the existence of 411bp and 416bp bands through double PCR amplification and electrophoresis detection. The molecular marker is significantly associated with salt tolerance phenotypes, the detection method is rapid, accurate and low in cost, the molecular marker can be used for brassica napus salt tolerance germplasm resource screening and molecular marker-assisted breeding, and the breeding efficiency is significantly improved.

The present application belongs to the technical field of molecular identification of new shrimp species, and particularly relates to a composition for identifying Neocaridina denticulata and Neocaridina zhoui and use thereof. The present application discloses a composition for identifying Neocaridina denticulata and Neocaridina zhoui, which comprises: a first specific primer pair with a nucleotide sequence as shown in SEQ ID NO: 3 and SEQ ID NO: 4; and a second specific primer pair with a nucleotide sequence as shown in SEQ ID NO: 5 and SEQ ID NO: 6. Based on the composition and the corresponding duplex PCR method provided by the present application, a 743 bp band specific to Neocaridina denticulata or a 601 bp band specific to Neocaridina zhoui can be amplified simultaneously in one reaction system, so that the two morphologically similar new shrimp species can be identified quickly, accurately and efficiently.

Groups of primers and probes for identifying eight kinds of animal-derived milk and milk products based on a double-tube duplex PCR and an application thereof are provided, which belongs to the technical field of milk product detection. The group of primers and probes of the present disclosure includes an internal reference forward primer and an internal reference reverse primer, a horse-derived forward primer and a horse-derived reverse primer, a donkey-derived forward primer and a donkey-derived reverse primer, a universal forward primer and a universal reverse primer, an internal reference probe, a buffalo-specific probe, a goat-specific probe, a sheep-specific probe, a horse-specific probe, a camel-specific probe, a yak-specific probe, a donkey-specific probe, and a cow-specific probe.

The invention relates to the technical field of molecular biology, and particularly discloses a method and a primer group for identifying Frankliniella occidentalis and Flavothrips tawaiiensis and application, the method comprises the following steps: extracting genome DNA of a to-be-detected sample, and using the primer group Sd-F, Sd-R, Fo-F and Fo-R to carry out dual PCR amplification; if only a band with the size of 254bp appears on the amplification product, determining that the sample to be detected is the thrips thealensis; if only a band with the size of 362bp appears on the amplification product, the sample to be detected is the Frankliniella occidentalis; if two bands with the lengths of 254bp and 362bp simultaneously appear on the amplification product, the sample to be detected is a mixed sample of the thrips theasali and the thrips occidentalis. The method provided by the invention can be used for quickly and accurately distinguishing and identifying the Frankliniella occidentalis and the Flavoniella rebaudiana, is high in specificity, and can be used for quickly identifying the important pests Frankliniella occidentalis and Flavoniella rebaudiana in a blueberry planting area in actual production.

The invention discloses a double PCR (Polymerase Chain Reaction) detection kit for trichina and toxoplasma gondii and application of the double PCR detection kit. The kit comprises upstream and downstream primer pairs for detecting trichina and toxoplasma gondii respectively, 2 * Taq Master Mix and non-enzyme water, the primer pair for detecting the trichina specifically aims at a complete sequence of a trichina T1 isolate ISS 003 ribosome small subunit gene, and the primer pair for detecting the toxoplasma gondii specifically aims at a toxoplasma gondii H66R isolate 529REP repetitive sequence region. The duplex PCR detection system disclosed by the invention is good in specificity, and does not generate cross reaction with other common polypide bodies; corresponding target fragments can be amplified when trichina and toxoplasma gondii are detected, and no amplified fragment appears when common pig parasites are detected, so that the specificity is high; the detection limit is 0.10 ng / mu L, so that the kit has relatively high sensitivity. In conclusion, the detection kit disclosed by the invention is high in sensitivity, strong in specificity, mild in reaction condition, short in reaction period and high in detection flux, and can be used for rapidly screening the toxoplasma gondii and trichina in meat products and live pigs in various scenes.

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.

The present invention relates to the technical field of molecular detection, and aims to provide a primer set for a duplex PCR method for simultaneously detecting Entamoeba suis and Entamoeba polecki, a method, and a use. The primer set comprises the following primers: species-specific primers for Entamoeba suis, namely forward primer E.suis F and reverse primer E.suis R, and species-specific primers for Entamoeba polecki, namely forward primer E.polecki F and reverse primer E.polecki R. The nucleotide sequences of E.suis F, E.suis R, E.polecki F, and E.polecki R are respectively shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No. 4. A duplex PCR detection system having high specificity, high sensitivity, time- and labor-saving properties, and easy operation is established. The primer combination and method can synchronously detect Entamoeba suis and Entamoeba polecki in a single reaction, can significantly reduce the cost and workload of detecting the two pathogens, and can detect positive DNA at a minimum concentration of 400 fg / μL. The method can be applied to the rapid detection and identification of infections caused by these two protozoa in the porcine intestinal tract.

The invention belongs to the technical field of pathogen detection, and particularly relates to a duplex PCR (polymerase chain reaction) method, primer group and kit for rapidly detecting influenza A virus H1N1 and porcine contagious pleuropneumonia, which comprises the following steps: extracting DNA (deoxyribonucleic acid) of a sample to be detected, preparing a duplex PCR reaction system, taking the DNA of the sample to be detected as a template, carrying out fluorescent PCR amplification by using the primer group, and when Ct is less than or equal to 35 and a specific amplification curve appears, determining that the sample to be detected is the influenza A virus H1N1 and porcine contagious pleuropneumonia. The result is positive; when Ct is larger than 35 and smaller than 40, repeated detection should be carried out, and if the result is still that Ct is larger than 35 and smaller than 40 and a specific amplification curve appears, the result is positive; when Ct is greater than or equal to 40 or Ct cannot be detected, the result is negative; wherein the FAM labeling probe and the primer aiming at the HA gene of the influenza A H1N1 virus are shown in the detailed content in a sequence table. The method is rapid and accurate in detection and high in sensitivity.

The invention discloses a microsatellite fluorescent multiplex PCR (polymerase chain reaction) method for paternity test of Chinese giant salamanders, and belongs to the technical field of bioengineering. The method comprises the following steps: extracting genomic DNA of a Chinese giant salamander individual Screening polymorphic microsatellite primers of the giant salamanders in China; optimization and amplification of Chinese giant salamander microsatellite multiple PCR conditions are carried out; and paternity testing. According to the invention, 11 screened highly-polymorphic microsatellite loci are utilized to establish a duplex PCR and triple fluorescent PCR system, a sequencer is used for typing and data reading, and individual identification and parent-child relationship analysis are carried out on Chinese giant salamander families; according to the invention, two or three sites can be detected through one PCR reaction, the PCR reaction procedure is optimized, and compared with single-site detection, the efficiency is improved by more than two times, and the cost is reduced to more than one half of the original cost; the establishment of the method provides a new technical means for germplasm identification, family management and enhancement and release effect evaluation of the Chinese giant salamanders.

The invention discloses a duplex PCR (Polymerase Chain Reaction) detection primer for flavobacterium psychrophilum and aeromonas salmonicida, and relates to a detection primer for flavobacterium psychrophilum and aeromonas salmonicida and application of the detection primer. The duplex PCR detection primer comprises an rpoC upstream primer / downstream primer and an fstA upstream primer / downstream primer, and the application of the duplex PCR detection primer of flavobacterium psychrophilum and aeromonas salmonicida in fish mixed pathogen detection is provided. When the rpoC and fstA primer combination is used for detecting flavobacterium psychrophilum and aeromonas salmonicida, the sensitivity of the rpoC and fstA primer combination can reach 1 * 10 <-4 > ng / mu L, and the sensitivity is good. The rainbow trout multiple bacterial diseases caused by flavobacterium psychrophilum and aeromonas salmonicida and the mixed infection of the rainbow trout multiple bacterial diseases have good application value in rapid identification of rainbow trout multiple bacterial diseases caused by flavobacterium psychrophilum and aeromonas salmonicida.

The present invention discloses an RPA-LFS detection primer-probe combination for encapsulated and non-encapsulated Haemophilus influenzae and its application; the primer-probe combination of the present invention has good specificity, no cross-amplification for other strains, can detect Haemophilus influenzae at 1 CFU / ul, and the detection sensitivity is not interfered by other pathogenic bacteria. The results of this study are consistent with those of duplex PCR. A total of 203 Haemophilus influenzae and 63 encapsulated Haemophilus influenzae were detected from 209 samples, with detection rates of 97.1% and 63.2% respectively. RPA-LFS has the same practical applicability as duplex PCR. This study successfully established an RPA-LFS detection method for detecting encapsulated and non-encapsulated Haemophilus influenzae based on the Omp6 and BexA genes, providing a solution for the subsequent rapid detection of Haemophilus influenzae and ensuring the timely diagnosis and antibiotic treatment of patients.

The embodiment of the invention discloses a microsatellite marker primer for identifying the genetic relationship of siniperca scherzeri, a kit, application and a method, the number of pairs of primers is ten, the ten pairs of primers are divided into five groups to be subjected to duplex PCR reaction, and low-cost, high-efficiency and standardized large-scale siniperca scherzeri paternity test and pedigree management can be achieved.