53 results about "Primer dimer" patented technology

The invention provides methods for simultaneously amplifying multiple nucleic acid regions of interest in one reaction volume as well as methods for selecting a library of primers for use in such amplification methods. The invention also provides library of primers with desirable characteristics, such as minimal formation of amplified primer dimers or other non-target amplicons.

The invention belongs to the field of nucleic acid detection and molecular biology, and particularly relates to a method and a kit for eliminating false positive results in nucleic acid amplification reaction. According to the method, after a nucleic acid amplification reaction (taking recombinase polymerase amplification, namely RPA, for example) is completed, a target amplification product is subjected to selective enzyme digestion by utilizing restriction endonuclease, non-target products (such as primer dimers and non-specific amplification products) are not cut, and meanwhile, the non-target products which are not subjected to enzyme digestion are removed by combining solid-phase separation, so that the target amplification product is obtained. Therefore, the false positive result is eliminated. The invention also discloses a kit containing the restriction enzyme. According to the method, the specificity and the signal-to-noise ratio of a nucleic acid amplification reaction, especially RPA, are remarkably improved, the cost is low, operation is easy, the method is compatible with an existing technical platform, the method is suitable for detection scenes such as clinical molecular diagnosis, environmental monitoring and food safety, and the problem of misjudgment caused by false positive signals is effectively avoided.

The invention provides methods for simultaneously amplifying multiple nucleic acid regions of interest in one reaction volume as well as methods for selecting a library of primers for use in such amplification methods. The invention also provides library of primers with desirable characteristics, such as minimal formation of amplified primer dimers or other non-target amplicons.

The invention belongs to the technical field of molecular biological detection, and particularly relates to a primer group for detecting salmonella pullorum based on an RAA-LFD technology and application of the primer group. The invention provides a primer group for detecting salmonella pullorum based on an RAA-LFD technology. The primer group comprises a forward primer, a reverse primer and a probe, the sequence of the forward primer is as shown in SEQ ID NO. 1; the sequence of the reverse primer is as shown in SEQ ID NO. 2; the sequence of the probe is based on SEQ ID NO.3, wherein the original base G of the 31st base of the SEQ ID NO.3 is replaced by an idSp group. According to the specific primer based on the RAA-LFD technology, the phenomenon of false positive caused by generation of primer dimers is avoided, the specificity is high, and the sensitivity is high. The primer group provided by the invention can be used for detecting salmonella pullorum and related strains, and only the salmonella pullorum is positive, so that the primer group shows good specificity.

The invention provides methods for simultaneously amplifying multiple nucleic acid regions of interest in one reaction volume as well as methods for selecting a library of primers for use in such amplification methods. The invention also provides library of primers with desirable characteristics, such as minimal formation of amplified primer dimers or other non-target amplicons.

The invention relates to the technical field of biology, and provides a primer group and a kit for simultaneously detecting a dengue virus and a Zika virus and application of the primer group, and the primer group is formed and comprises a primer pair 1 aiming at the dengue virus, a primer pair 2 aiming at the Zika virus and a universal LAMP (Loop-Mediated Isothermal Amplification) primer pair. When in use, target virus single-stranded RNA is used as a bridge molecule, under the catalysis of Splint R ligase, adjacent DNA probes with specific sequences are guided and connected, and a double-ring (dumbbell-shaped) structure product containing a general cyclization primer sequence is directly formed. The two key technical breakthroughs of forming a universal double-ring primer through target RNA mediated probe connection and outputting a high-specificity signal dependent on RNase H2 are realized; a dengue virus and Zika virus isothermal nucleic acid amplification detection platform which is simple and convenient to operate, rapid in reaction, high in sensitivity, strong in specificity (capable of effectively avoiding interference of primer dimers and non-specific dyes) and suitable for multiple detection is constructed, and compared with conventional fluorescent quantitative PCR, the dengue virus and Zika virus isothermal nucleic acid amplification detection platform has remarkable advantages.

The present invention provides a method for simultaneously amplifying multiple target nucleic acid regions in a single reaction volume, and a method for selecting a primer library to be used in such amplification. Furthermore, the present invention provides a primer library having desired properties, such as minimal formation of amplification primer dimers or other non-target amplification products. [Solution] For example, a method for amplifying a target gene locus in a nucleic acid sample is provided, comprising the steps of (a) contacting the nucleic acid sample with a test primer library that simultaneously hybridizes to at least 1,000 different target gene loci to generate a reaction mixture, and (b) subjecting the reaction mixture to primer extension reaction conditions to generate an amplification product containing a target amplification product.

The present invention relates to a method for amplifying at least three target nucleic acid molecules with reduced primer dimer formation in a multiplex amplification reaction. The method of present invention can inhibit primer dimer formation and hence generation of nonspecific amplification products in an effective manner in a multiplex amplification reaction for at least three target nucleic acid molecules.

The application discloses a DPO RT-PCR primer group for synchronously detecting five citrus viruses, a detection method, a kit and application of the DPO RT-PCR primer group, and sequences of the primer group are shown as SEQ ID NO. 1-SEQ ID NO. 10. The application designs specific DPO RT-PCR primers for citrus tristeza virus, citrus tatter leaf virus, citrus yellow vein clearing virus and citrus leaf mottle virus, and the detection primers can effectively remove primer dimer interference, and the five viruses can be detected from samples at the same time. The detection method is simple and efficient, and only needs to use a common PCR instrument and an electrophoresis instrument to detect, and has the characteristics of rapidness, economy, high accuracy and high sensitivity, and can be used for large-scale screening of field citrus plants and epidemic prevention detection before seedling transportation, and provides technical support for healthy seedling detection and field screening of diseases.

The application discloses a method for constructing a tag sequence-containing vector library by short oligonucleotides and application thereof. The method needs two oligonucleotides, i.e. a customized tag primer and a universal primer which can be used for all library constructions. The middle of the tag primer is a tag sequence, and both sides are annealing sequences. The universal primer has sequences complementary to the annealing sequences of the tag primer on both sides, and a modified base which can be recognized as a base damage by a host microorganism in the middle. The tag primer library is annealed with an equal amount of the universal primer to obtain primer dimers. The primer dimers have base-paired DNA double strands on both sides, and are cohesive ends. The middle of the primer dimers is an unpaired omega loop structure, and the double strands of the omega loop contain the tag information carried by the tag primer and the modified base carried by the universal primer respectively. The primer dimers are connected with linearized vectors, and then are transformed into E. coli, so that the region of the modified base of the omega loop is replaced by the tag sequence through the base excision repair mechanism of the endogenous cells.

The invention provides methods for simultaneously amplifying multiple nucleic acid regions of interest in one reaction volume as well as methods for selecting a library of primers for use in such amplification methods. The invention also provides library of primers with desirable characteristics, such as minimal formation of amplified primer dimers or other non-target amplicons.

An object of the present invention is to provide a design method, a manufacturing method, a design device, a design program, and a recording medium of a primer for amplicon methylation sequence analysis, which can improve a design success rate of the primer while suppressing the formation rate of a primer dimer extremely low.The present invention is a primer design method for amplicon methylation sequence analysis, including a primer sequence determination step of selecting one or more primer candidate sequence pairs related to a predetermined target site from one or more primer candidate sequences, calculating a local alignment score between predetermined primer sequences, and adopting and determining a primer candidate sequence pair having a score equal to or less than a predetermined threshold value as a forward primer sequence and a reverse primer sequence for amplifying a region including the predetermined target site.

Multiplex PCR amplification may be improved by using primers with exonuclease-resistant bonds or nucleotides to reduce the formation of primer dimers. Multiplex PCR-based assays may be improved by using a load-balanced set of primers with different proportions of primers in the set to provide balanced amplicon read depth.

The invention provides methods for simultaneously amplifying multiple nucleic acid regions of interest in one reaction volume as well as methods for selecting a library of primers for use in such amplification methods. The invention also provides library of primers with desirable characteristics, such as minimal formation of amplified primer dimers or other non-target amplicons.

The invention provides a quadruple fluorescent quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection method for pathogenic viruses of porcine intestinal diseases, namely a quadruple fluorescent quantitative RT-PCR detection method suitable for detecting diarrhea of pigs in China, and the quadruple fluorescent quantitative RT-PCR detection method can be used for detecting PEDV (Porcine Epidemic Diarrhea), TGEV (Transmissible Gastroenteritis), aPoRVA (Porcine Rotavirus) and PDCoV (Porcine Delta Coronavirus) in one PCR amplification reaction. According to the invention, rapid and high-precision detection of PEDV, TGEV, PoRVA and PDCoV is realized by a real-time fluorescent quantitative PCR technology, and the kit has important significance in prevention of rapid propagation and timely purification treatment of PEDV, TGEV, PoRVA and PDCoV in the future, and has a good application prospect. The method solves the problem that spectrum overlapping of different probes in multiple channels affects signal crossing, avoids false positive, can reduce generation of primer dimers in amplification, and solves the technical problem that segmented annealing is needed due to large annealing temperature difference.

The invention discloses a primer dimer scoring method, system, equipment and medium and application in primer optimization, and belongs to the technical field of primer design. The invention provides a novel scoring method and system for a primer dimer, which can remarkably improve the identification capability of potential risks of the dimer. Furthermore, by means of the dimer scoring method and system, the primer pool can be dynamically optimized, and the number of formed dimers is remarkably reduced. After the targeted sequencing primer pool is dynamically optimized by using the method and the system, the proportion of dimer sequencing data is reduced by 39%, and the sensitivity of a detection system is greatly improved.

The present invention relates to the field of biotechnology, and particularly to a DNA extraction reagent for a Rana spinosa InDel molecular marker sample and a preparation method thereof. The DNA extraction reagent for a Rana spinosa InDel molecular marker sample provided by the present invention comprises 60-70 parts of a lysate, 8-12 parts of a protease mixture, 10-15 parts of an impurity remover, 6-9 parts of a DNA precipitant, and 10-15 parts of a washing solution. Each component is prepared through specific steps. DNA is extracted using the reagent, and through steps such as sample pretreatment, lysis, impurity removal, precipitation, washing, and dissolution, impurities can be effectively removed, thereby improving DNA purity, concentration, and integrity. PCR amplification has a good effect, and the product band is single and clear, free of primer dimers and non-specific amplification. This greatly improves the accuracy and reliability of Rana spinosa InDel molecular marker detection, and promotes Rana spinosa molecular genetics research and related industries.

The invention discloses a primer group and a kit for simultaneously detecting a panda aleutian virus and a panda canine distemper virus Asia type 6 and application of the primer group and the kit, and belongs to the technical field of virus detection of endangered wild animals. The invention provides a primer group and a kit for specifically recognizing the aleutian disease virus and the canine distemper virus Asia 6 type of the panda, and detection can be completed within 20 minutes under the condition of constant temperature of 39 DEG C; according to the present invention, the method has characteristics of rapid detection, good specificity, high sensitivity and the like, does not produce the cross reaction with other common pathogens, does not produce the false positive phenomenon due to the generation of the primer dimer, has characteristics of rapid detection, good specificity, high sensitivity and the like, and has characteristics of simple and easy operation, and on-site detection without special detection personnel; the method is rapid and accurate in detection, has a good application effect in preliminary application, and provides a method for rapid diagnosis, virus traceability, epidemic disease monitoring and early warning of a wild animal epidemic disease source and the like in zoos and wild animal rescue institutions.

The present application relates to the technical field of sex chromosome STR identification, and particularly relates to an X chromosome STR locus composite amplification primer group, a 9-color fluorescent label composite amplification kit and application thereof.The composite amplification primer group comprises 45 pairs of specific amplification primer pairs, the 45 pairs of specific amplification primer pairs include 44 X chromosome STR loci and 1 pair of specific amplification primer pairs of a gender locus AMEL, and the nucleotide sequences of the primer pairs in the composite amplification primer group are shown as SEQ ID NO.1-90.The specificity of each primer in the composite amplification primer group is relatively strong, no primer dimer or interaction is generated during composite PCR amplification, the amplification efficiency difference of each pair of primers is relatively small, and the identification sensitivity is relatively high.The composite amplification primer group provides a molecular basis and core necessary reagent for simultaneously detecting 44 X-STR loci at a time.

This invention discloses a primer set, reagent kit, and their applications for the simultaneous detection of Aleutian virus and Canine Distemper Virus Asia 6 (Asia 6) in red pandas, belonging to the field of endangered wildlife virus detection technology. This invention provides a primer set and reagent kit that specifically identifies Aleutian virus and Canine Distemper Virus Asia 6 in red pandas. Detection can be completed within 20 minutes under constant temperature conditions of 39℃; it does not cross-react with other common pathogens and does not produce false positives due to primer dimer formation. In other words, it features rapid detection, high specificity, and high sensitivity. The implementation process is simple and easy to operate, requiring no specialized testing personnel for on-site testing. The detection is rapid and accurate, and initial applications have shown good results, providing a method for rapid diagnosis, virus tracing, wildlife disease monitoring, and early warning in zoos and wildlife rescue institutions.

Multiplex PCR-based assays may be improved by using a load-balanced set of primers with different proportions of primers in the set to provide balanced amplicon read depth. The multiplex PCR amplification in such assays may be improved by using primers with exonuclease-resistant bonds or nucleotides to reduce the formation of primer dimers.

The application relates to a primer dimer scoring method, a primer dimer scoring system, a primer dimer scoring device and a primer dimer scoring medium and application in primer optimization, and belongs to the technical field of primer design. The application provides a new scoring method and system for primer dimers, which can significantly improve the identification ability of potential risks of dimers. Further, by using the dimer scoring method and system, the primer pool can be dynamically optimized, and the number of formed dimers can be significantly reduced. After the primer pool for targeted sequencing is dynamically optimized by using the method and system, the proportion of dimer sequencing data is reduced by 39%, and the sensitivity of the detection system is greatly improved.

Multiplex PCR-based assays may be improved by using a load-balanced set of primers with different proportions of primers in the set to provide balanced amplicon read depth. The multiplex PCR amplification in such assays may be improved by using primers with exonuclease-resistant bonds or nucleotides to reduce the formation of primer dimers.

The application provides a primer probe set, a method and a kit for detecting nucleic acid, the primer probe set comprises a first primer, a second primer and a probe; wherein the probe comprises a primer anchor region and a probe signal detection region, the primer anchor region is located at the 5' end of the probe signal detection region, and the probe is modified with a detection label; the first primer comprises a probe anchor region, a connection sequence and a target sequence binding region from 5' end to 3' end in sequence, and the second primer comprises a primer signal detection region, a connection sequence and a target sequence binding region from 5' end to 3' end in sequence. When the primer probe set is used for nucleic acid detection, the phenomenon of primer dimerization can be reduced, and false positive results caused by primer dimers or other non-specific amplification can be effectively avoided. The nucleic acid detection method can realize single-tube multiplex detection, has low requirements on the length of the target sequence and high detection sensitivity.

Methods as well as related compositions and kits for recombinase-mediated nucleic acid amplification, such as recombinase-polymerase amplification (RPA), of a nucleic acid template using at least one blocked primer that contains a 5′ domain, at least one nucleotide that is cleavable by an RNase H enzyme, a 3′ domain, wherein the primer is not extendable by a polymerase, and wherein the 3′ domain has a length of 7-100 nucleotides, for example 10-30 nucleotides. These methods and the use of a blocked primer reduce or eliminate non-specific amplification products, such as primer dimers, which are generated in RPA reactions.

The invention relates to application of SLS in preparation of a product capable of improving LAMP detection performance, and belongs to the technical field of biology. The invention provides an application of sodium lauroyl sarcosinate (SLS) in preparation of a product capable of improving LAMP (loop-mediated isothermal amplification) detection performance. On one hand, the SLS has a target specificity optimization effect on detection of targets such as the monkey pox virus in an LAMP reaction system, and by adjusting the ion environment of the LAMP reaction system, the SLS can improve the specific amplification efficiency of the monkey pox virus target by 20% or above while completely inhibiting non-specific amplification; on the other hand, the SLS has the effect of improving the sensitivity and the detection rate on detection of targets such as the monkey pox virus in an LAMP reaction system, and under the low template concentration, the SLS can improve the detection rate of the monkey pox virus targets from 60% to 100% by inhibiting formation of primer dimers. Therefore, the SLS has a promising application prospect in LAMP-based nucleic acid detection.

The invention provides methods for simultaneously amplifying multiple nucleic acid regions of interest in one reaction volume as well as methods for selecting a library of primers for use in such amplification methods. The invention also provides library of primers with desirable characteristics, such as minimal formation of amplified primer dimers or other non-target amplicons.

Provided in the present invention is a method for detecting and differentiating multiple different targets in a single-tube PCR reaction by using single-tube ultra-multiplex PCR. The method can avoid false positive signals generated by a primer dimer when multiple specific primers are present. Further provided in the present invention are a primer and probe for the single-tube super-multiplex PCR, a reaction system, and a multiple-target detection kit containing the primer, the probe and the reaction system.

The application provides primers and probes for rapid detection of tobacco target spot pathogen, application, kit and a rapid detection method of tobacco target spot pathogen, and relates to the technical field of tobacco prevention and treatment. The primers and probes for rapid detection of tobacco target spot pathogen are screened out through test optimization, have high specificity for the tobacco target spot pathogen, the amplification product is a single band, and there is no non-specific amplification and obvious primer dimer. The primers and probes for rapid detection of tobacco target spot pathogen are used for RPA amplification of the target gene of the tobacco target spot pathogen, and the lateral flow chromatographic test strip is used for visual detection of the target gene, and has the advantages of high sensitivity, good specificity and short operation time.

The invention relates to a method for detecting DNA modification, in particular to a construction method of a DNA library with modified nucleotide, a detection method, a kit set and application. According to the scheme provided by the invention, the necessity of designing degenerate primers is avoided, the design difficulty of multiple primers is greatly reduced, the specificity of multiple PCR amplification is improved, and the formation of primer dimers is reduced.