75results about How to "Avoid non-specific amplification" patented technology

The invention relates to a respiratory tract common pathogen multiple PT-PCR combined gene chip detection kit. The kit detects one or more of 22 respiratory tract common pathogens by the usage of multiple specific conservative degenerate primer combination and probe combination and provided with endogenous control, positive control and negative control at the same time. According to the detection kit, the coverage rate for a high mutant pathogen subject sequence is increased, the problem of nonspecific cross reaction between the multiple primers and the probe is avoided, one single reaction system can detect more than 20 respiratory tract common pathogens simultaneously, and a detection tool which is simple and sensitive, rapid and large in flux and capable of carrying out multi-index parallel detection is provided for respiratory tract common pathogen detection.

The invention discloses a method for rapidly detecting chicken, duck and pig blood components in a blood jelly. The method comprises the following steps: taking the blood jelly as a material to extract DNA (Deoxyribose Nucleic Acid); carrying out fluorescence quantitative PCR (Polymerase Chain Reaction) by using a TaqMan probe and a specific primer; applying ABI7500SofwareSDS1.4 to carry out analysis processing on an experiment result; taking amplification with a Ct value less than 36 as a positive result of the detection. The method disclosed by the invention is a multi-TaqMan probe real-time fluorescence PCR method for simultaneously detecting the chicken blood component, the duck blood component and the pig blood component in the blood jelly so as to avoid non-specific amplification; various animal original components can be identified simultaneously; the detection efficiency and the result accuracy are improved greatly; the detection lower limit is 0.15ng and the sensitivity reaches 1%; the method has good specificity and can be used for simultaneously detecting and quantifying DNA components of duck blood, pig blood and chicken blood in the blood jelly, thus exploring a new way for identifying the animal original components in food and having very good practicability.

The invention discloses a detection kit and a method for copy number of spinal muscular atrophy pathogenic gene SMN1 based on a real-time fluorescence quantitative PCR (Polymerase Chain Reaction) technique. The detection kit comprises specific primers SMN1-F (SEQ ID NO.1) and SMN1-R (SEQ ID NO.2) modified by NO.7 exon locked nucleic acid of a pair of target genes SMN1, an SMN1 detection probe (SEQID NO.3), an SMN2 gene closed probe SMN2-B (SEQ ID NO.4) modified by SMN-P locked nucleic acid and 3'end C3Spacer, gene primers ALB-F (SEQ ID NO.5) and ALB-R (SEQ ID NO.6) of a reference gene ALB andan ALB detection probe ALB-P (SEQ ID NO.7). The amplification specificity and amplification efficiency of the SMN1 gene are enhanced by using locked nucleic acid modification; a stable reaction system for double amplification of the target genes and the reference gene is established by using a multi-PCR principle and optimizing reaction systems and conditions, and further quantitative accuracy and reliability of the copy number of the SMN1 can be ensured; good repeatability and high operability are obtained.

The invention provides a detection method for deletion and insertion mutation of L367fs*46 (c.1179-1230del) and K385fs*4(c.1234-1235insTTGTC) of the ninth exon of a CALR (Calreticulin) gene. Universal amplification primers are designed according to deletion and insertion mutation areas of the ninth exon of the CALR gene, specific detection fluorescence probes are designed according to amplified fragments, real-time fluorescence quantification PCR (polymerase chain reaction) is performed on a to-be-detected sample, and deletion and insertion mutation of the CALR gene are identified through multiple single-tube reactions. The invention further provides applications of the universal amplification primers and the specific detection fluorescence probes in preparation of myeloproliferative neoplasm detection reagents as well as a kit for detecting myeloproliferative neoplasms.

The invention discloses a LAMP detection kit and application, and belongs to the technical field of biology. The kit provided by the invention comprises a LAMP amplification reaction system and a colloidal gold test strip, wherein the LAMP amplification reaction system comprises a LAMP primer group and LAMP amplification reaction liquid; the LAMP primer group comprises inner primers FIP and BIP, outer primers F3 and B3, and loop primers LF and LB; the LAMP amplification reaction liquid contains Taq SSB proteins, Bst DNA polymerase, Bst buffer, dNTPs, Mg<2+>, a template and ddH2O. The kit has the advantages of easiness in operation, low detection cost, short detection time, high specificity and high sensitivity; by adopting the kit, the primers can be prevented from generating polymers, the generation of primer dimers is reduced or prevented, the generation of false positive is prevented, and meanwhile the amplification efficiency of a LAMP reaction is increased.

The invention belongs to the technical field of molecular biological detection, and provides a method for detecting and identifying six pairs of PCR (polymerase chain reaction) primers of six kinds of Listeria bacteria from an environment respectively. The PCR primer pairs are designed according to a specific target gene iap (inhibitor of apoptosis protein) of the Listeria bacteria, the target gene is high in stability and specificity, and the six kinds of Listeria bacteria existing in the environment can be rapidly and accurately identified by virtue of a reasonable PCR system and a gel electrophoresis detection means. The detection method comprises the following steps of (1) extracting a sample DNA (deoxyribonucleic acid), and performing PCR amplification by adopting different primer pairs respectively; (2) judging whether a sample to be detected contains the Listeria bacteria or not according to the length of a PCR amplification product fragment. According to the method, the six kinds of Listeria bacteria in the environment and a food sample can be rapidly, accurately and sensitively detected and identified, so that the efficiency is improved, time is saved, and moreover, the detection cost is lowered.

The invention discloses a method for detecting the activity of DNA methyltransferase based on single-ribonucleotide repair-mediated ligation-dependent cycling signal amplification. The method comprises the following steps: (1) adding a Dam MTase acted hairpin substrate to a resection reaction buffer solution, and carrying out a cleavage reaction to produce a 24-nt product; and (2) adding the cleavage product to an amplification reaction buffer solution, carrying out a cyclic linkage-dependent strand displacement amplification reaction to cyclically generate an enhanced fluorescent signal, anddetecting the intensity of the fluorescent signal to determine the activity of the Dam MTase. The method can effectively inhibit nonspecific amplification independent of a target and a template / primer, so the background signal is greatly reduced, and the index amplification of the signal is finally achieved. The method has a high sensitivity and a high specificity, and the lower detection limit can reach 4.8 * 10<-6> U / mL.

The invention discloses a stem-loop structured combined probe and application thereof. The probe is constituted by two DNA sequences of stem-loop structures; from 5' terminals to 3' terminals, probe I and probe II sequentially include stem-loop zones and detection object recognition zones, and are applicable to telomerase activity, RNA and DNA detection and telomerase inhibitor screening; when a detection object is telomerase, the detection object recognition zone of the probe I is a telomerase substrate sequence, and the detection object recognition zone of the probe II is a sequence which is complementary with 8-40 bases in a telomerase extension sequence; and when the detection object is RNA or DNA, the detection object recognition zone of the probe I is a sequence which is complementary with 8-40 bases, close to the 3' terminal, of to-be-detected RAN or DNA, and the detection object recognition zone of the probe II is a sequence which is equal to 8-40 bases, close to the 5' terminal, of the to-be-detected RAN or DNA. The combined probe disclosed by the invention is unnecessary to make use of fluorescence labeling and radioactive element labeling, and the combined probe, with the specific stem-loop structure, provides a specific amplification template for a nucleic acid amplification reaction, so that rapid signal amplification under constant temperature is achieved; and the combined probe is high in sensitivity and amplification efficiency.

The invention discloses a ring mediated cascade amplification strategy used for detecting DNA transmethylase activity in a high sensitivity manner. A long-stem ring probe is designed on the basis of strand displacement amplification and exponential type rolling circle amplification. The probe comprises a methylation locus, a long-stem part and a ring part, wherein the methylation locus is used for identifying DNA transmethylase, the long-stem part is used for guaranteeing the stability of the probe, and the ring part is used for triggering subsequent amplification; the ring part and a small part of stem part are used as the triggering strands for subsequent signal output. The probe is characterized in that the triggering strands are completely sealed in the ring part of the probe by the long-stem part, and non-specific amplification caused by probe leakage is avoided; the long-stem ring probe is methylated by the DNA transmethylase and then cut by restriction endonuclease to produce the triggering strands; under the synergic effect of polymerase and the endonuclease, the produced triggering strands trigger strand displacement amplification to produce a large amount of primers; the produced primers trigger exponential type rolling circle amplification to synthesize a large amount of G-tetraploid sequences, and the sequences interact with dye to obtain enhanced fluorescent signals.

The invention discloses primers, a micro-fluidic chip and a system for detecting swine fever viruses, and application of the primers, wherein the primers are a primer group with the following sequences: P1:AGGGACTAGCCGTAGTGG, P2: AGGTCGTACCCCCATCAC, p3: GGTGAGCTTCTGCTCATGTCGA-AGCTCCCTGGGTGGTCTA, and P4: CGAGATGCTATGTGGACGAGGG-TGTGAGTTCACCCTAGCGA. According to the invention, the multiple defects ofLAMP isothermal amplification are thoroughly overcome, the detection effects of simple operation, rapid result output, multiple target points, synchronization and integration are realized, the characteristics of high sensitivity and high specificity are achieved, and a plurality of different indexes such as the swine fever virus and the like of the same sample or swine fever virus indexes of a plurality of samples can be detected at one time.

The invention discloses a method for detecting a plurality of food allergens in a sample, which comprises: (1) preparing a visible thin film sensor chip, wherein a plurality of specific probes corresponding to the DNA sequences of the plurality of allergens are fixed on the surface of a chip substrate respectively; (2) performing multiplex polymerase chain reaction (PCR) amplification on the DNA sequence of a test sample by using the plurality of pairs of specific upstream and downstream primers corresponding to the DNA sequences of the plurality of allergens, wherein the 5' terminal of the downstream primer has biotin bonded by a covalent bond; (3) crossing the product of the PCR amplification in the step (2) with the probes on the visible chip, and washing; (4) crossing horseradish peroxidase-marked biotin with the chip, and eluting uncrossed chains; (5) processing and developing the chip by using a horseradish peroxidase substrate; and (6), determining if the sample has the allergens and the types of the allergens according to the generation condition of a visible signal and the position of the probe corresponding to the generated visible signal. The method can quickly and synchronously detect if the sample has various allergens with high throughput.

The invention discloses a thermostatic strand displacement amplification technology. According to the technology, after heating denaturation is conducted on a target DNA sequence, a pair of primers containing RNA sequences are used for hybridizing with a template DNA single chain, the primers are extended under the action of strand displacement DNA polymerase, a combined sequence of target DNA andRNA containing an identification site of endoribonuclease is generated, the identification site is cut by the hydrolyzation of thermal resistant endoribonuclease to form viscous termini, the primersare connected with the viscous termini, a 3' terminus is extended under the action of the strand displacement DNA polymerase and replaces another DNA strand, a displaced strand is sequentially turnedinto target sources of another amplification reaction after the displaced strand is hybridized with the primers, the steps are constantly repeated in a reaction system, and the target DNA sequence isincreased in exponential rate. The technology is convenience, sensitive, rapid and good in specificity, the detecting lower limit is DNA with a plurality of gene copy numbers, the technical detect ofthermostatic amplification in the prior art is overcome, and the technology can be widely applied to the fields of clinical medicine, medical jurisprudence, animal and plant quarantine inspection andthe like.

The invention discloses a folding primer multiple PCR malaria molecule diagnostic kit and a detection method thereof. The kit is composed of a magnesium-containing template buffer and a magnesium-free single combination reagent, and comprises a complete set of reagents for DNA preparation and a folding primer PCR reaction. The folding primer takes plasmodium Cyt b gene as a target gene, designing is carried out on plasmodium and specific site, sensitivity and singularity of four plasmodium detected by folding primer PCR can be obviously increased, and reagent and test operation can be simultaneously simplified. The kit of the present invention is employed for detection, a traditional reagent complex operation of nested-set / multiple PCR is simplified to a single reagent and a single tube for one step amplification reaction, four plasmodium individual or mixing infection can be detected and identified simultaneously, minimum of detected monocontamination can reach or approach 1 plasmodium / muml blood; and four protozoon can be simultaneously detected and accurately identified when the mixing infection is high, and low density difference is 100 times. The diagnostic kit and the detection method is a reliable means for definite diagnosis and discriminating of malaria in hospitals and laboratory in province and city.

The invention provides an identifying and diagnosing method for African swine fever virus gene I type and African swine fever virus gene II type based on a probe-oriented recombinase-mediated isothermal amplification technique. The method can be used for quickly diagnosing and distinguishing the African swine fever virus gene I type and the African swine fever virus gene II type. The invention relates to a probe for detecting specific difference sites of different genotypes based on the probe-oriented recombinase-mediated isothermal amplification method. According to the probe, based on the probe used in a recombinase-mediated isothermal amplification method, tetrahydrofuran is used for replacing a backward base of a site or a backward base of a specific difference site complementary site,excision enzymes III are subjected to enzymolysis until cutting of a tetrahydrofuran site is finished, a cutoff fluorophore modified base produces fluorescence signals in a system, and remaining probe parts exert the effect of a downstream primer. The probe can simplify necessary downstream primer components in the detection course, and a downstream primer and the probe are combined for use, so that the possibility that the probe and the primer generate non-specific amplification is reduced.

The invention discloses a high-efficiency high-sensitivity universal shell type fluorescent reverse transcription-polymerase chain reaction (RT-PCR) detection method of bird flu virus and a detection kit. The detection method is a method in which two sets of polymerase chain reaction (PCR) primers (one pair of outer primers and one pair of inner primers) are utilized to carry out two rounds of PCR amplification, namely, the products of the outer primers subjected to the PCR amplification are used as templates of the inner primers to be subjected to the PCR amplification, the bonding sites of the inner primers and the template DNAs are positioned at the inner sides of the DNA fragments amplified by the outer primers, and shell type PCR is extremely effective for reducing or eliminating nonspecific amplification and improving sensitivity. Compared with the fact that the temperate with an extremely low concentration (one or multiple copies) is difficult to detect by using the common detection method, the efficiency and fidelity of amplification can be greatly improved by using the detection method in the invention; and the detection method disclosed by the invention is significantly effective for the amplification of extremely trace target genes in environmental samples, is extraordinarily beneficial to the amplification of the trace temperate of the bird flu virus in a fish farming water body, and can fully meeting the requirements of the sensitivity and specificity of the bird flu virus detection in fishpond farming water.

The invention relates to a construction method of a magnetic microsphere DNA probe for detecting miRNA molecules as well as a product and an application of the magnetic microsphere DNA probe, which belong to the technical field of molecular probes. The invention provides the magnetic microsphere DNA probe for detecting miRNA molecules, and the linear amplification of detection signals can be achieved on the premise that the number of initial miRNAs in a sample is kept unchanged. As the probe does not need PCR in the detection process, non-specific amplification is avoided, and the detection specificity is improved. Compared with a traditional separation method, the magnetic beads are used for separating complex components of a biochemical sample, separation and enrichment can be carried out at the same time, the separation speed and the enrichment efficiency are effectively improved, and meanwhile the sensitivity of analysis and detection is greatly improved.

The invention relates to a quantitative PCR method adopting a dye EvaGreen and dual HotStar polymerases. The quantitative PCR method is characterized by comprising the following steps that 1, a PCR reaction system, including a sample to be tested, a forward primer and a reverse primer complementary with templates to be tested in the sample to be tested, the fluorescent dye EvaGreen and the dual HotStar polymerases including a HotStar Taq DNA polymerase and an Anti Taq DNA polymerase, is established; 2, PCR amplification is conducted on the templates to be tested in the PCR reaction system to form a double-stranded DNA-EvaGreen fluorescent dye compound; 3, a fluorescence value of the PCR reaction system is detected during or after the PCR amplification to determine the number of amplification products so as to determine the number of templates to be tested. The quantitative PCR method has the advantages of being high in accuracy, high in efficiency, high in flexibility, high in specificity and low in price.

The invention provides a probe and kit for detecting an SNP (Single Nucleotide Polymorphism) site by utilizing a recombinase mediated isothermal amplification method based on probe guidance and belongs to the technical field of SNP site detection. According to the probe for detecting the SNP site by utilizing the recombinase mediated isothermal amplification method based on the probe guidance, onthe basis of the probe for the recombinase mediated isothermal amplification method, the probe is used for carrying out enzymolysis on a next-site basic group of a tetrahydrofuran replacement SNP siteor a next-site basic group of an SNP site complementary site by an excision enzyme III until tetrahydrofuran site cutting is finished; a cut-off fluorescence group modified basic group generates a fluorescence signal in a system and residual probes are partially used as downstream primers. A simplified detection process of the probe needs a downstream primer component so that nonspecific amplification between the probe and the primer is reduced. The invention provides the kit for detecting the SNP site and the kit comprises an upstream primer and the probe. The kit can be used for detecting various SNP sites.

The invention belongs to the technical field of gene engineering, and discloses a primer combination for HLA (human leukocyte antigen) genotyping, a kit containing the primer combination and a method for HLA genotyping. Thus, the sequence-specific HLA genotyping (GSA-SBT) technique is quick, simple, accurate and visual. The primer combination can be synchronously used for amplification and sequencing with the commercialized kit, thereby lowering the experimentation cost. Besides, the specific design of mismatched bases are introduced to the 3' terminal of the primer, thereby enhancing the specificity of the primer amplification and ensuring the accuracy of the typing result.

The invention discloses a solid-phase PCR kit for gene detection of hypertension medication. The solid-phase PCR kit comprises hot-start DNA polymerase, uracil-N-glycosylase, a PCR reaction buffer solution, dNTPs and a primer. The invention further discloses a detection method adopting the solid-phase PCR kit for gene detection of hypertension medication. The detection method comprises the following specific steps: collecting a sample, extracting nucleic acid, wherein the concentration is not lower than 10ng / ul, and the purity is 1.8-2.0; by taking the nucleic acid sample as a template, carrying out solid-phase PCR amplification reaction by adopting the prepared solid-phase PCR kit for gene detection of hypertension medication, wherein the 25ul PCR reaction system comprises 23ul the solid-phase PCR kit for gene detection of hypertension medication and 2ul the nucleic acid template; carrying out electrophoresis analysis on the PCR product by adopting 1.0% agarose gel; and carrying out result judgment. The solid-phase PCR kit has the advantages that the operation is convenient, the detection efficiency is high, etc.

The invention belongs to the technical field of whole-course heat start Taq enzyme preparation, and discloses thermostable Taq enzyme temperature control affinity ligand, and preparation method and application thereof. The amino acid sequence of the ligand is as shown in SEQ ID NO:1, the ligand is thermostable under the condition that the temperature is less than or equal to 96 DEG C, the closed or opened Taq enzyme activity is reversible when the temperature is less than or equal to 96 DEG C, the ligand can take part in heat start of the Taq enzyme in the whole course in the whole PCR circulation, Taq enzyme heat start is conducted only when a primer and a template are scrupulously matched in each circulation process, non-specific amplification and generation of a primer dimer are avoided, reaction specificity and sensitivity are improved, the DNA polymerase chain reaction result has few impurities and low noise, and the fidelity is perfect; meanwhile, the ligand adopts a short peptide structure, has smaller molecular weight and smaller PCR interference compared with those of an antibody, is not like single chain DNA which is easy to degrade, and the whole-course heat start Taq enzyme product prepared by the ligand has higher stability.

The invention discloses a reagent kit for multi-channel fluorescent PCR detection of the polymorphism of IL28B gene loci. The reagent kit can specifically detect the polymorphism of the IL28B gene loci rs12979860 and rs8099917, the two loci are simultaneously detected in one PCR reaction pipe, and the specific genotype is made clear and definite. Besides, operation is easy, convenient and fast, the result is objective and high in controllability, efficiency is high, cost is low, contamination is avoided through closed-pipe operation, and the reagent kit is suitable for a multi-channel fluorescent PCR instrument and is a powerful product for clinically detecting the polymorphism of the IL28B gene loci.

The invention discloses a molecular detection method for Edrus deodara (Roxb) Lobd blight and primers therefor, and establishes a set of quick, high-sensitivity, stable and reliable molecular detection technology for detecting the Edrus deodara (Roxb) Lobd blight. The method has the advantages of accuracy, quickness, simplicity in operation and the like, can identify pathogens in an early stage of contagious disease, can be referentially applied to inspection and quarantine and field investigation of imported and exported plants and plant products, and has an important significance for controlling the Edrus deodara (Roxb) Lobd blight to spread in China. Meanwhile, the establishment of the system provides technical guidance and theoretical basis for detecting other pathogenic bacteria.

The invention discloses a nucleic acid composition for detecting HPVs (Human Papilloma Viruses) as well as application and a kit of the nucleic acid composition, and relates to the technical field ofin-vitro diagnosis of virus nucleic acid detection. The nucleic acid composition for detecting the HPV comprises primers which are shown in SEQ ID NO.1 to 6 and primers which are shown in SEQ ID NO.7to 9. The nucleic acid composition disclosed by the invention is capable of simultaneously detecting HPV 16 and HPV 18 and parting the specificity of the HPV 16 and the HPV 18, meanwhile, quality control can also be carried out on a detecting process through an internal control beta-globin gene detection result, false negatives can be reduced, and the nucleic acid composition has the characteristics of high sensitivity and strong specificity.

The invention discloses a linked gene detection kit for a potato yellow dwarf virus and a detecting method. The kit comprises one set of specific primers and fluorescence probes for a potato yellow dwarf virus RdRp gene, one set of specific primers and fluorescence probes for a potato yellow dwarf virus NP gene, RT Buffer, a random primer, a RNA enzyme inhibiting factor, dNTPs, reverse transcriptase, TaqMan PCR mix, a positive control sample and a negative control sample. The invention further discloses a reaction system, a reaction condition and result determination for performing real-time fluorescent quantitative RT-PCR detection on the potato yellow dwarf virus through a linked gene. According to the experiment, the method can effectively detect potato yellow dwarf viruses of differentplant systems, has the advantages of short detection time, high specificity, high sensitivity, simple and convenient operation, safe and high flux, and is very suitable for quick detection on entry and exit port quarantine and the potato yellow dwarf virus on agricultural production.

The invention belongs to the technical field of nucleic acid detection, and particularly relates to a primer probe combination, a detection kit and an application of the detection kit. A first Y-typeprimer and a second Y-type primer are designed, the first Y-type primer can be partially complementary with a specific primer of a dengue virus, and a second Y-type primer can be partially complementary with a specific primer of a Zika virus to form a Y-type structure for preventing starting of a non-specific extension reaction. The problem of insufficient detection specificity caused by high similarity of genomic sequences of the dengue virus and the Zika virus is solved, so that the specificity and the accuracy in the detection process of the dengue virus and / or the Zika virus are improved,and good application prospect and market value are achieved.

The invention discloses a method for development of specific PCR markers of SNP loci related to an F-type three-line hybrid wheat male sterility gene, wherein the method comprises the steps: (1) carrying out multi-generation sister crossing on wheat, dividing the obtained selfing lines into a maintainer line with high self-fruitful rate, a maintainer line with medium self-fertility rate, and a stable sterile line with self-fruitful rate low than 5%, wherein the three lines are identical in other biological characteristics except having fertility difference; (2) carrying out library-building and sequencing of the three samples; (3) searching for SNP variations between mitochondrial and cell nucleus genomes of the three samples; and (4) aiming at mitochondrial and cell nucleus genome SNP loci obtained after identification of the sterile line and the maintainer lines with different fruitfulness rates, detecting, designing and developing restriction endonuclease amplified polymorphism sequence (CAPS) markers or competitive allele specific PCR (KASP) markers. The invention also discloses relevant SNP loci molecular markers and a detection method thereof.