54results about How to "Consistent amplification efficiency" patented technology

The invention relates to a multiplex-PCR three-round amplification method, a primer and a kit thereof and application of the method in establishment of next-generation sequencing platform libraries. In the first two rounds of a PCR, a specific primer with the low concentration is consumed as much as possible, so that the differences of different amplicons are reduced and the homogeneity of multiplex-PCR products is improved; when the third round of the PCR is performed, adapter primers carrying different bar code sequences are added to mark different templates, the different adapter primers carry same universal amplification primers, therefore, it is guaranteed that the amplification efficiency of the different templates is consistent, and the differences of different template amplification products are reduced. Accordingly, not only is the problem that the homogeneity of ordinary multiplex-PCR amplification is poor solved, but also establishment of the sequencing libraries can be quickly and conveniently completed.

The invention belongs to the field of nucleic acid detection and discloses a method for semi-quantitatively detecting pathogenic nucleic acid by adding internal control nucleic acid. Corresponding internal control is added in the whole process of extracting and amplifying target nucleic acid and testing by using a test paper, so that the internal control and a target segment are parallelly operated, and the semi-quantitative detection is performed finally through color development and intensity contrast of three strips, namely a detection line, an internal control line and a quality control line on the test paper. In the method, in the whole process of processing the target nucleic acid, the corresponding internal control is taken as a positive contrast, and false negative results due to links such as extraction, amplification or sample application errors are avoided in the processing of detecting by using the test paper. Meanwhile, by comparing color development intensity of the internal control line and a sample line and introducing the semi-quantitative function on the basis of the qualitative function of the immunochromatographic test paper to estimate the copy number of tested samples, the detection results are more detailed, accurate and reliable. The method has the advantages of convenient and quick operation and capacity of meeting the actual clinical requirement.

The invention belongs to the field of forensic medicine, and discloses a microhaplotype genetic marker for forensic detection and a kit thereof. The invention provides a genetic marker combination consisting of 21 microhaplotypes located on autosomal chromosomes. The genetic marker combination can be effectively applied to forensic detection. Compared with the traditional detection kit, the microhaplotype genetic marker for forensic detection and the kit thereof provided by the invention have high sensitivity, accurate detection result and high applicability in individual identification, can realize individual identification of human biological samples in a relatively short period of time, and wins valuable time for solving of forensic cases.

The invention discloses a dual real-time fluorescence PCR (Polymerase Chain Reaction) detection primer pair, probes, a kit and a detection method for type 1 and type 2 porcine circovirus. The dual real-time fluorescence PCR detection primer pair for type 1 and type 2 porcine circovirus is characterized in that the nucleotide sequence of the type 1 specific porcine circovirus is as shown in SEQ ID NO: 9 and SEQ ID NO: 11. The probe comprises the nucleotide sequence as shown in SEQ ID NO: 14. The nucleotide sequence of the type 2 specific porcine circovirus is as shown in SEQ ID NO: 2 and SEQ ID NO: 6. The probe comprises the nucleotide sequence as shown in SEQ ID NO: 7. The kit provided by the invention has the characteristics of rapidness, simpleness and convenience, strong specificity, high sensitivity and good reliability, and can be used for analyzing samples in batches at the same time, and differentiating whether the sample is type 1 porcine circovirus infection or type 2 porcine circovirus infection or not by reaction at one time, or common infection of type 1 and type 2 porcine circovirus infection, thereby, providing a powerful technical support for monitoring and preventing porcine circovirus epidemic situation. The kit has good application prospect.

The invention relates to functional genomics and a gene expression detection and analysis technology, in particular to a kit for quantitative evaluation for long-term recurrent risk of colorectal cancer. The kit is characterized by consisting of 18 pairs of primers, 18 specific taqman fluorescent probes, 10* reverse transcription-polymerase chain reaction (RT-PCR) buffer solution, deoxyribonucleoside triphosphate (dNTP) mixed liquid, reverse transcriptase, deoxyribose nucleic acid (DNA) polymerase, 10* PCR buffer solution and ribonucleic acid (RNA) enzyme inhibitor. By adopting the self-designed and optimized RT primers and integrating a RT-PCR technology and a taqman real-time fluorescent quantitative PCR technology, the kit is simple and rapid in operation, more stable in diction results and lower in detection cost.

The invention discloses a yellow crucian carp high-density SNP (single nucleotide polymorphism) marker screening method, which includes extracting genomic DNA, constructing a RAD library and high-throughput sequencing, filtering original sequence data, and detecting and screening SNP information sites; adding circular DNA and chitin in the process of extracting the genomic DNA to effectively improve the screening accuracy of SNP markers; and adding chitin fluid into a PCR (Polymerase Chain Reaction) amplification system to improve the reaction sensitivity of PCR amplification and help to obtain the complete high-density SNP sites. The high-density SNP sites obtained by the method can be applied to analyze the genetic diversity level, population genetic structure and the like of the yellowcrucian carp population. The method for obtaining the high-density SNP marker sites of the yellow crucian carp has the advantages of simplicity, high efficiency, high SNP marker screening accuracy, good repeatability, low cost and the like.

The invention tries to establish a multi-cow-disease detecting method based on a GeXP multi-gene expression genetic analysis system. The problems that when various cow diseases are detected, a serological method is low in sensitivity, only a single pathogen can be detected through a conventional PCR, and a gene chip method is high in cost are solved. Three cow disease RNA positive sample reference products are established. A 10-fold continuous diluted reference product of the reference product RNA serves as a detecting object, and 100 copies can be detected in terms of sensitivity; no cross reaction or false negative can happen between the cow diseases and other cow diseases, 600 actual samples are detected, and the requirements for monitoring and screening a large number of cow diseases are met.

GeXP multiple rapid detection primers for detection of bluetongue virus, bovine viral diarrhea virus and foot-and-mouth disease virus and a detection method are provided; the detection method for various cow diseases based on a GeXP multiple gene expression genetic analysis system is intended to be built up; the problems that when detection of the various cow diseases, a serological method has low sensitivity, conventional PCR only detects single pathogens and a gene chip method has relatively high cost are solved; three cow disease DNA positive sample standard plasmids are constructed; a reference obtained by 10-time continuous dilution of the plasmids DNA is used as a detection object, and the detection sensitivity is 10-100 copies; no cross reactions with other cow diseases happen, and no false negative results exist; the number of detected actual simples is 600, and monitoring screening requirements of various diseases of a lot of cows are met.

The invention discloses a kit for simultaneously detecting multiple cat pathogens as well as a detection method and application of the kit. A specific primer group and a probe group are obtained through design, so that rapid and accurate detection and identification on feline plague viruses, feline calicivirus, feline infectious peritonitis viruses, feline toxoplasma gondii, feline herpes viruses, feline chlamydia and feline mycoplasma in one reaction are realized at the same time. The detection kit obtained on the basis of the design is high in specificity and sensitivity, the feline pathogens with the concentration of 1.7 * 10 <2> copies / mL can be effectively detected, repeated detection is not needed, the actual application accuracy is high, and the detection kit can be widely applied to epidemic situation monitoring, prevention and control of feline virus infection.

This invention involves the method of measuring Lamivudine's medicine-resisting HBV DNA and reagent box. Correlated with two kinds of main sudden change genes (YMDD sudden change is for YVDD and YIDD) design peculiar sudden change measure primer and virus peculiar measure primer and the public related probe, or design peculiar sudden change measure probe and virus peculiar measuring probe to conduct the real-time measure to the same sample respectively in three PCR tubes through the same PCR procedure. According to the D value of the Ct value in reaction tube 1, V reaction tube with the Ct value of the C reaction tube (í¸Ctú¡i=Ctú¡iú¡Ctú¡cú¼í¸Ctú¡v=Ctú¡vú¡Ctú¡c) to judge the YVDD and YIDD medicine-resisting sudden change situation of HBV in clinical sample. Advantages: stable reagent box performance, high sensitivity and so on.

The invention discloses a screening method for adaptability SNP loci of Japanese eels. The method comprises the steps of extraction of a genome DNA, construction and sequencing of an RAD library, RADdata output and quality control and detection and screening of the SNP loci. The screening method for the adaptability SNP loci of the Japanese eels has the advantages that 37,700 SNP loci screened out through the method are widely distributed in the whole genome range of a Japanese eel sketch and totally contained in 10,710 contigs, each contig contains 3.5 SNP loci on average, genome informationresources of the Japanese eels which are endangered species are further supplemented and completed, and the important basis is provided for correlational research of the population genetic structureand population historical dynamic condition of the Japanese eels.

The invention aims to provide a reagent assisting in identifying a sowbane mosaic virus and application thereof. The reagent provided by the invention comprises a specific primer consisting of deoxyribonucleic acid (DNA) shown as a sequence 1 in a sequence table, DNA shown as a sequence 2 in the sequence table and DNA shown as a sequence 3 in the sequence table. The reagent can also comprise a specific probe of which the nucleotide sequence is shown as a sequence 4 in the sequence table. The sowbane mosaic virus is an important quarantine harmful pest in China. A method for detecting the SoMV by a nested-RT-Realtime polymerase chain reaction (PCR) is established by three nested PCR primers and a TagMan probe. In the method, a nested PCR and real-time fluorescent PCR technology are combined organically; two sets of PCR systems which consist of three primers and a probe are verified mutually, so that the accuracy of a result is enhanced effectively; and detection sensitivity is enhanced effectively by the real-time fluorescent PCR technology. The method is correct, sensitive, simple, convenient and rapid, and the lower detection limit is up to 0.4 fg / mu l of plant total RNA.

The invention discloses a dual fluorescent probe primer combination for identifying African swine fever type I strains and type II strains, a kit and application of the dual fluorescent probe primer combination and the kit, and belongs to the technical field of molecular biology. The dual fluorescent probe primer combination comprises a primer probe group for detecting an African swine fever virus type I strain and a primer probe group for detecting a universal P72 gene of the African swine fever virus. According to the invention, genomes are analyzed and compared in a conserved region of the African swine fever virus, the gene B117L is finally screened out, fragments with great difference between a type I virus strain and a type II virus strain are selected, and probe primer design and reaction condition optimization are carried out, so that the amplification efficiencies of the two virus genes are similar, and a kit convenient for diagnosis is developed. The kit can detect the African swine fever virus I type infection, the African swine fever virus II type infection or co-infection of the two viruses at one time, has the advantages of high sensitivity, strong specificity and good repeatability, and provides technical support for prevention and control of the African swine fever in a pig farm.

The invention discloses GeXP quick detection multi-primers and a detection method for detecting akabane viruses, bovine viral diarrhea viruses and blue tongue viruses. The GeXP quick detection multi-primers and the detection method have the advantages that the detection method is created for detecting diversified epidemic diseases of cows on the basis of GeXP multi-gene expression genetic analysis systems, so that problems that serological methods are low in sensitivity, only single pathogens can be detected by the aid of conventional PCR (polymerase chain reaction), gene chip methods are high in cost and the like when diversified epidemic diseases of cows are about to be detected at present can be solved, and reference of RNA (ribonucleic acid) positive samples of three epidemic diseases of the cows is created; 10-times continuous dilution reference of reference RNA is used as a detection object, and the detection sensitivity can reach 100 copies; cross reaction between the GeXP quick detection multi-primers and the other epidemic diseases of the cows and false-negative results can be prevented; monitoring and screening requirements on large quantities of diversified epidemic diseases of cows can be met by 600 actual detection samples.

The invention relates to the field of bioengineering and particularly relates to primers, a method and a kit for detecting the expression amount of a chicken FAM134B gene. The primers are shown in the formulas of SEQ ID NO: 1 to 6. The method comprises extracting total RNAs from chicken tissue, carrying out reverse transcription to obtain cDNA and carrying out fluorescent quantitative PCR on specific primers of an amplification target gene and a reference gene. The kit is composed of the specific primers shown in the formulas of SEQ ID NO: 1 to 6, SYBR Premix Ex Taq II (Tli RNaseH Plus) (2x), ROX Reference Dye II (50x) and sterile water. The detection method is simple and fast, can measure the gene expression amount of the FAM134B gene in chicken and can study the expression pattern of the FAM134B gene in various tissues of the chicken.

The invention aims at providing a reagent for assisting in identifying potato viruses A and application thereof. The reagent provided by the invention comprises a specific primer consisting of DNAs shown as in a sequence 1, a sequence 2, a sequence 4 and a sequence 5 of a sequence table as well as a probe A shown as in a sequence 3 and a probe B shown as in a sequence 6. The potato viruses A are important quarantine harmful organisms in China. The invention provides two sets of PCR (Polymerase Chain Reaction) primer probe compositions and establishes a method for detecting PVA (Plyvinyl Aetate) by dual primer probes of -RT-Realtime PCR. By adopting a real-time fluorescent PCR technology, the method effectively improves the sensitivity of the detection. Two sets of primer probes with consistent grain efficiency are mutually verified and determined so as to effectively improve the accuracy of the result and achieve stronger operability in actual detection. The method is accurate, sensitive, simple, convenient and quick; and the detected low limits of the potato viruses can respectively reach 0.5fg / mul of total RNA (Ribonucleic Acid) of plant.

The invention discloses GeXP multi-primers and a method for detecting akabane viruses, bovine viral diarrhea viruses and foot and mouth disease viruses. The GeXP multi-primers and the method have the advantages that the method is created for detecting diversified epidemic diseases of cows on the basis of GeXP multi-gene expression genetic analysis systems, so that problems that serological methods are low in sensitivity, only single pathogens can be detected by the aid of conventional PCR (polymerase chain reaction), gene chip methods are high in cost and the like when diversified epidemic diseases of cows are about to be detected at present can be solved, and reference of RNA (ribonucleic acid) positive samples of three epidemic diseases of the cows is created; 10-times continuous dilution reference of reference RNA is used as a detection object, and the detection sensitivity can reach 10-100 copies; cross reaction between the GeXP multi-primers and the other epidemic diseases of the cows and false-negative results can be prevented; monitoring and screening requirements on large quantities of epidemic diseases of cows can be met by 600 actual detection samples.

The invention provides a kit and a usage method thereof. The kit comprises one or more pairs of 16 upstream and downstream primer pairs for amplifying STR loci, namely, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, CSF1PO, FGA, TH01, TPOX, vWA, Penta E, Penta D and Amelogenin, wherein the sequences of the 16 pairs of upstream and downstream primers are shown as SEQ ID NO:1 to SEQ ID NO:32. By adopting the kit and the usage method provided by the invention, the related 16 pairs of upstream and downstream primers have the advantages of high specificity and high amplification efficiency, and genotyping of the 16 STR loci can be performed rapidly and accurately.

The invention provides a reagent for assistant identification of carnation ringspot viruses and an application thereof. The reagent comprises a specific primer formed from DNAs shown in a sequence 1, a sequence 2, a sequence 4 and a sequence 5 in a sequence table, a probe A shown in a sequence 3 and a probe B shown in a sequence 6. The carnation ringspot viruses are major quarantine harmful organisms in China. The invention provides two PCR (Polymerase Chain Reaction) combinations of primers and probes, and creates a method for using double real-time PCR combinations of primers and probes to detect the carnation ringspot viruses. The method adopts the real-time fluorescence PCR technology, and effectively increases the sensitivity of detection. The two sets of combinations of primers and probes, which have consistent amplification efficiency, are authenticated by each other, thus, the accuracy of results is increased, and the method has strong operability in actual detection. The method is accurate, sensitive, simple, convenient and quick, and has the low detection limit reaching 0.46 fg / mul of total RANs of plants.

The invention discloses a detection kit for charcot marie tooth disease related PMP22 gene copy number variation. The detection kit comprises a 10*PCR buffer solution, a 4*GC solution, a PCR primer mixed solution, TaqDNA polymerase, a probe primer mixed solution, a DNA diluent, a 10*Taq connecting buffer solution, Taq ligase, 20m MEDTA, dNTP (2.5mM) and MgCl2 (25mM). The kit and the system for detecting PMP22 gene copy number variation on the basis of a CNVplex-high flux connecting probe amplification technology (HLPA) disclosed by the invention are mainly used for detecting the charcot marietooth disease caused by the PMP22 gene deletion or repeated mutation so as to acquire the correct copy number of each target locus of the PMP22 gene. The detection kit has the advantages of quick detection, high accuracy, high repeatability, high resolution ratio, and the like.

This study intends to establish a detection method for multiple dairy cow diseases based on the GeXP multiple gene expression genetic analysis system to solve the problems of low sensitivity of serological methods in the detection of various dairy cow diseases, conventional PCR can only detect a single pathogen, and the high cost of gene chip methods. Construct 3 kinds of dairy cow disease RNA positive sample reference products; 10-fold serial dilution of the reference sample RNA is used as the detection object, and the sensitivity is to detect 100 copies; there is no cross-reaction with other dairy cow diseases, and there is no false negative; 600 actual samples are tested , to meet the monitoring and screening requirements of a large number of dairy cows for various diseases.

The invention aims at providing a reagent for assisting in identifying potato viruses V and application thereof. The reagent provided by the invention comprises a specific primer consisting of DNAs shown as in a sequence 1, a sequence 2 and a sequence 3 of a sequence table as well as a probe of the nucleotide sequence shown as in a sequence 4. The potato viruses V are important quarantine harmful organisms in China. The invention establishes a method for detecting PVA (Plyvinyl Aetate) by heminested-RT-Realtime PCR(Polymerase Chain Reaction) by using three PCR primers and one TaqMan probe. The method is organically combined with the nested PCR and the real-time fluorescent PCR technology; two sets of PCR systems formed by three primers are mutually verified to effectively improve the accuracy of the result; and a fluorescent probe effectively improves the sensitivity of the detection. Proved by experimental results, the method is accurate, sensitive, simple, convenient and quick; and the detected low limits of the potato viruses can respectively reach 0.5fg / mul of total RNA (Ribonucleic Acid) of plant.

The invention provides a construction method of a microhaplotype multiplex amplification system in mixed sample analysis and related haplotype frequency. The invention provides a group of genetic marker combinations, in total of 18 microhaplotypes and rs numbers of the 18 microhaplotypes . According to the 18 microhaplotype amplification primers, a microhaplotype composite amplification system formixed sample analysis is constructed. Also provided herein is a test kit comprising amplification primers of the aforementioned 18 microhaplotypes. The microhaplotype multiplex amplification system provided by the invention can be used for carrying out forensic medicine identification on a mixed test material, and is high in sensitivity and strong in identification capability.

The invention provides a micro nucleic acid joint testing method and kit. Multiple target micro nucleic acids are captured by hybridization of single-stranded template probes; excessive single-stranded template probes are removed by enzyme digestion of mung bean nuclease; a template probe forming double strands with the target micro nucleic acids is obtained by purification; then PCR amplificationtesting is carried out; and a positive amplification result indicates the presence of the target micro nucleic acid. According to the method and the kit, the PCR pretreatment steps of micro nucleic acid testing are reduced to three steps; the flux is increased to 4 times of a traditional PCR method, and the interference of genome DNA, a primary precursor, a secondary precursor and a double-stranded precursor is avoided, thus being conducive to improving and promoting popularization and application of micro nucleic acid testing.

The invention aims at providing a reagent for assisting the identification of arabis mosaic virus and an application thereof. The reagent provided by the invention comprises a specific primer consisting of DNA (Deoxyribonucleic Acid) shown in a sequence 1 in a sequence table, DNA shown in a sequence 2 in the sequence table, and DNA shown in a sequence 3 in the sequence table. The reagent also comprises a specific probe shown in a sequence 4 in a nucleotide sequence table. The arabis mosaic virus is an important quarantine pest in China, and a method for detecting ArMV by semi-nested-RT (Reverse Transcriptase)-Realtime PCR (Polymerase Chain Reaction) is established by utilizing three nested type PCR primers and a TaqMan Probe. According to the method, the nested PCR technology and the real-time fluorescence PCR technology are organically combined; two PCR systems formed by the three primers and the probe are verified with each other, so that the result accuracy is effectively improved; the detection flexibility is effectively improved by the real-time fluorescence PCR technology; and the accuracy, the flexibility, the simplicity, the convenience and the rapidness are realized, and the minimum detection limit can reach 0.5fg / mul plant total RNA (Ribonucleic Acid).

The invention aims at establishing a detecting method for multiple dairy-cow epidemic diseases based on a GeXP-multiple-genetic-expression genetic analysis system. The problems that when the multiple dairy-cow epidemic diseases are detected, the sensitivity of the serology method is low, only a single pathogene is detected through conventional PCR, and the cost of the gene chip method is high are solved. Four dairy-cow epidemic-disease RNA-positive-sample reference products are built; ten-fold continuous dilution reference products of RNA of the reference products serve as detected objects, and the sensitivity is detected to 10-100 copies; the dairy-cow epidemic disease is free of cross reaction with other dairy-cow epidemic diseases, and false negative is avoided; 600 parts of practical samples are detected, and the monitoring screening requirements of the multiple epidemic diseases of a large number of dairy cows are met.

The invention provides a micro-haplotype composite amplification system in the analysis of degraded sample materials, a construction method thereof and related haplotype frequencies. The present invention provides a group of genetic marker compositions totaling 23 micro-haplotypes and their rs numbers. According to the amplification primers of 23 micro-haplotypes, a complex amplification system of micro-haplotypes for the analysis of degraded samples was constructed. Also provided herein is a kit comprising amplification primers for the aforementioned 23 micro-haplotypes. The micro-haplotype complex amplification system provided by the invention can carry out forensic identification on degraded samples, and has high sensitivity and strong identification ability.

The invention discloses a quadruple quantitative fluorescent probe primer combination, a kit and an identification method for identifying an African swine fever wild virus and a vaccine strain, and belongs to the technical field of molecular biology. The quadruple quantitative fluorescent probe primer combination for identifying the African swine fever wild virus and the vaccine strain comprises: (a) a first primer probe combination as shown in SEQ ID NO:1-SEQ ID NO:3; (b) a second primer probe combination as shown in SEQ ID NO:4 to SEQ ID NO:6; (c) a third primer probe combination as shown in SEQ ID NO:7-SEQ ID NO:9; and (d) a fourth primer probe combination as shown in SEQ ID NO:10 to SEQ ID NO:12. The kit and the identification method can simultaneously identify four genes of the African swine fever wild strain and the vaccine strain, primers of different genes do not interfere with each other, and the kit and the identification method have the characteristics of high sensitivity, strong specificity, good repeatability and good stability.

The invention is aimed at providing a reagent assisting to identify a tobacco ringspot virus (TRSV) and an application thereof. The reagent provided by the invention comprises the component of: a specific primer composed of a DNA expressed in a sequence 1 in a sequence table, a DNA expressed in a sequence 2 in the sequence table and a DNA expressed in a sequence 3 in the sequence table. The reagent further comprises a specific probe with a nucleotide sequence as shown in a sequence 4 in a nucleotide sequence table. The tobacco ringspot virus is an important quarantine pest in our country. According to the reagent and the application, a method for detecting the TRSV through semi-nested-RT-Realtime PCR is created through three nested PCR primers and a TaqMan probe. In the method, the nested PCR technology and the real-time fluorescence PCR technology are organically combined; two PCR systems respectively formed by the three primers and the one probe mutually verify to effectively improve the accuracy of the result; and the real-time fluorescence PCR technology is used for effectively improving the detection sensitivity. The method is accurate, sensitive, simple, convenient and quick, and the detection minimum can be up to 4 fg / ul of total plant RNA.

The invention provides a microhaplotype composite amplification system in degradation sample analysis, a construction method thereof and related haplotype frequency. The invention provides 23 microhaplotypes in total and rs numbers thereof in one group of genetic marker compositions. According to amplification primers of the 23 microhaplotypes, a microhaplotype composite amplification system for analyzing degradation samples is constructed. The invention also provides a kit comprising the amplification primers of the 23 microhaplotypes. The microhaplotype composite amplification system provided by the invention can be used for forensic identification of the degradation samples, and is high in sensitivity and strong in recognition capability.