55 results about "Gene expansion" patented technology

The invention relates to a group of specific amplification primers for mitochondrial genes of procambarus clarkia and application thereof, and belongs to the technical field of molecular biology, and the mitochondrial genes of the procambarus clarkia comprise cytochrome c oxidase I subunit genes and cytochrome b genes. The specific amplification primer of the CO I gene comprises a CO I gene upstream primer and a CO I gene downstream primer, wherein the base sequences of the CO I gene upstream primer and the CO I gene downstream primer are respectively shown as SEQ NO.1 and SEQ NO.2; the specific amplification primer of the Cytb gene comprises a Cytb gene upstream primer and a Cytb gene downstream primer of which the base sequences are respectively as shown in SEQ NO.3 and SEQ NO.4. The invention further discloses a kit for detecting the Cytb gene. According to the amplification method of the mitochondrial gene of the procambarus clarkia, PCR (Polymerase Chain Reaction) amplification is carried out on a DNA (Deoxyribonucleic Acid) template solution of the procambarus clarkia by adopting amplification primers of a mitochondrial COI gene and a Cytb gene of the procambarus clarkia. The procambarus clarkia mitochondrial gene specific amplification primer has high specificity, a single target gene can be stably amplified, the length of the obtained procambarus clarkia mitochondrial COI gene segment reaches about 1000 bp, and the length of the obtained procambarus clarkia mitochondrial Cytb gene segment reaches about 800 bp.

The present invention discloses an application of an insect virus FHV RNA1 replicon in exogenous gene amplification, wherein the application amplifies the exogenous gene at the mRNA level by utilizing the autonomous replication ability of the insect virus FHV RNA1 replicon; the present invention discloses a plasmid system based on the insect virus FHV RNA1 replicon and a method for constructing the plasmid system; the present invention also discloses a method and application of the plasmid system for expressing proteins in cells, wherein the plasmid system is used to obtain a cloning vector after double enzyme digestion, and after homologous recombination with an exogenous gene, the animal or plant protein is expressed in the cell.

The invention discloses an HLA (human leukocyte antigen) gene amplification primer based on a nanopore sequencing platform and a typing method, and relates to the technical field of gene engineering, the amplification primer comprises an HLA-A gene amplification primer pair: a forward primer HLA-A-F5; a reverse primer HLA-A-R5; an HLA-B gene amplification primer pair: a forward primer HLA-B-F3; a reverse primer HLA-B-R3; the HLA-DQB1 gene amplification primer pair comprises a forward primer HLA-DQB1-F7, a reverse primer HLA-DQB1-F7 and a reverse primer HLA-DQB1-F7; a reverse primer of HLA-DQB1-R7; the HLA-DRB1 gene amplification primer pair comprises a forward primer HLA-DRB1-F1, a reverse primer HLA-DRB1-F2, a reverse primer HLA-DRB1 A reverse primer (HLA-DRB1-R1); the HLA-DRB3 gene amplification primer pair comprises a forward primer HLA-DRB3-F6, a reverse primer HLA-DRB3-F6, a reverse primer A reverse primer (HLA-DRB3-R8); by designing the specific amplification primer group and combining with the nanopore sequencing length reading technology, the problems of insufficient resolution and the like of the existing typing technology are solved, the typing accuracy and resolution are improved, and the performance is superior to that of the traditional technology; meanwhile, the process is optimized, the limitation of long detection period and the like is broken through, the whole process is rapidly completed, the technical threshold and detection expenditure are reduced, high efficiency, convenience and economy are considered, and support is provided for clinical popularization and scientific research application.

The invention belongs to the technical field of microbiological detection, and particularly relates to a Klebsiella pneumoniae MLST typing primer group and method based on a hospital localized microbiological mNGS platform. The primer group comprises primer pairs for amplifying an rpoB gene, an infB gene, a pgi gene, a phoE gene, a gapA gene, an mdh gene and a tonB gene, and corresponding nucleotide sequences are as shown in SEQ ID NO: 1-14. According to the technical characteristics of short sequencing read length (50 bp) and single-ended sequencing of a next-generation sequencing platform in a hospital, the primer group for Klebsiella pneumoniae MLST housekeeping gene amplification is redesigned, and amplicons are adaptive to the sequencing working principle of the existing next-generation sequencing platform in the hospital by optimizing the length and position of the amplicons. By utilizing the primer group, the kit or the method, the genetic relationship between hospital infection pathogenic bacteria can be quickly and accurately judged (within 24 hours), the timeliness and scientificity of infection prevention and control are remarkably improved, and the primer group, the kit or the method have a wide clinical application prospect.

The present application relates to a kind of triplex-forming oligonucleotides (TFOs) sequence and its pharmaceutical application, especially to a kind of triplex-forming oligonucleotides sequence and its pharmaceutical application for inhibiting MDM2 and MDM4 gene amplification.The triplex-forming oligonucleotides sequence provided by the present application can form triplex structure locally in the amplified MDM2 / MDM4 gene of tumor cell, cause its replication stress, finally lead to the apoptosis of tumor cell.The triplex-forming oligonucleotides sequence provided by the present application and the medicine prepared using the sequence are aimed at the MDM2 / MDM4 amplification gene sequence of tumor rather than protein, have good drug resistance, and have no effect on normal cell without MDM2 / MDM4 gene amplification, have excellent safety, thus have good therapeutic effect on the tumor with MDM2 / MDM4 gene amplification.

The application discloses application of a peach fruit hexokinase PpHXK3 gene in preparation of a plant cold-resistant product, and is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO. 2; a cloning method of PpHXK3 comprises the following steps: extracting total RNA of peach fruit and reversely transcribing the total RNA into cDNA as a template, and designing upper and lower amplification primers according to a gene sequence of PpHXK3; finally, a PpHXK3 gene amplification product is obtained through PCR amplification; the application of PpHXK3 in preparation of polygalacturonase PpPG21 inhibitors and / or polygalacturonase PpPG23 inhibitors, and the application of PpHXK3 in preparation of peach fruit cold-resistant products and heterologous plant cold-resistant products are also provided; and the advantages are that the decomposition of pectin is reduced, the integrity of a cell wall structure and the hardness of a fruit are maintained, the fruit has higher cold resistance, and cold damage in a storage and transportation process is reduced.

The invention relates to the technical field of gene detection, in particular to a rapid shrimp iridovirus detection primer group based on an RPA-CRISPR / Cas12a system, application and a kit. The primer group comprises an iridovirus constant-temperature gene amplification primer and a specific recognition amplification product target sequence probe; wherein the iridovirus constant-temperature gene amplification primer, the specific recognition amplification product target sequence CRISPR probe and the ssDNA reporter probe are recorded in the specification. An isothermal amplification primer and CrRNA are designed based on the shrimp red color virus gene, the target gene is amplified through the isothermal amplification technology, Cas12a is guided by the CrRNA to accurately recognize and activate trans-cleavage, a test strip report probe is synchronously split, and the result is interpreted by naked eyes within 30 min by virtue of the visual advantage of an immune test strip. The whole process does not need large instruments, the operation is simplified, the cost is low, and a visual and efficient solution is provided for on-site rapid diagnosis.

The invention discloses an SNP (Single Nucleotide Polymorphism) site, a DNA (Deoxyribose Nucleic Acid) fragment, a molecular marker primer and a method for identifying the color of cerasus humilis flesh, the SNP site is located at the 422nd basic group site of a cerasus humilis DFR gene, the polymorphism is T / G, the SNP site of red flesh is T, and the SNP site of yellow flesh is G. The nucleotide sequence of the DNA fragment is as shown in SEQ ID NO.1 or SEQ ID NO.2, and the molecular marker primer comprises a forward primer as shown in SEQ ID NO.3 and a reverse primer as shown in SEQ ID NO.4. The molecular marker primer has the advantages that the nucleotide sequence of the DNA fragment is as shown in SEQ ID NO.1 or SEQ ID NO.2; the method comprises the following steps: (1) collecting a to-be-detected cerasus humilis plant tissue sample and extracting total RNA (Ribonucleic Acid); (2) carrying out reverse transcription on the total RNA to obtain cDNA (complementary deoxyribonucleic acid); step (3), amplifying the cerasus humilis DFR gene by using the molecular marker primer by taking the cDNA as a template; (4) purifying and sequencing an amplification product; and (5) judging whether the pulp of the to-be-detected cerasus humilis sample is red or yellow according to a sequencing result. The technical problems that a traditional cerasus humilis pulp color identification method is long in period and not beneficial to popularization can be solved.

1. The name of the design product: Gradient gene amplification instrument (gradient PCR instrument). 2. The use of the design product: for gene amplification reaction. 3. The design points of the design product: in the combination of shape and pattern. 4. The picture or photo that best indicates the design points: perspective view.

The invention discloses application of a peach fruit hexokinase PpHXK3 gene in preparation of a plant cold-resistant product. The peach fruit hexokinase PpHXK3 gene is characterized in that a nucleotide sequence is shown as SEQ ID NO.1, and an amino acid sequence is shown as SEQ ID NO.2; the cloning method of the PpHXK3 comprises the following steps: extracting total RNA (Ribonucleic Acid) of peach fruits, reversely transcribing the total RNA into cDNA (Complementary Deoxyribonucleic Acid) as a template, designing upstream and downstream amplification primers according to a PpHXK3 gene sequence, and finally, carrying out PCR (Polymerase Chain Reaction) amplification to obtain a PpHXK3 gene amplification product. The invention further provides application of the PpHXK3 in preparation of a polygalacturonase PpPG21 inhibitor and / or a polygalacturonase PpPG23 inhibitor and application of the PpHXK3 in preparation of peach fruit cold-resistant products and heterologous plant cold-resistant products, and the PpHXK3 has the advantages that decomposition of pectin is reduced, integrity of a cell wall structure and hardness of fruits are maintained, so that the fruits have high cold resistance, and the yield of the fruits is increased. And the cold damage in the storage and transportation process is reduced.

The utility model discloses a gene amplification instrument with a test tube easy to take out, which comprises a machine body provided with a test tank and hinged with a tank cover; the heating module is provided with a plurality of test tube insertion holes, and the plurality of test tube insertion holes are arranged in a multi-row and multi-column matrix; the plurality of up-and-down lifting structures are arranged corresponding to the plurality of rows of test tube insertion holes, and the up-and-down lifting structures can move up and down relative to the machine body; and the lifting driving structure drives the multiple up-down lifting structures to move up and down, so that the multiple up-down lifting structures are arranged in a stepped fall mode. When the groove cover is turned upwards, the lifting driving structure drives the multiple up-down lifting structures to be arranged in a stepped fall mode. The vertical lifting structure can drive the test tubes to ascend in the ascending process, so that the multiple rows of test tubes are arranged in a stepped fall mode, and by means of the arrangement, under the condition that the multiple test tubes are tightly arranged, workers can easily take out the test tubes, and the test tubes can be taken out more conveniently.

This automated gene testing device enables accurate and rapid determination of gene amplification reactions within a reaction vessel through precise dispensing operations controlled by the rotation of a microfluidic chip and precise heating operations controlled by the temperature of the microfluidic chip. It allows for simple and rapid genetic testing of multiple viruses and other pathogens at the genetic level, even in on-site settings, without requiring special expertise or skills. [Solution] A gene amplification reaction is performed using a disc-shaped test chip equipped with a microfluidic centrifugal dispensing function, comprising a liquid delivery unit, a heating unit, an imaging unit, a display unit, and a control unit. The test chip comprises an injection reservoir set with a liquid sample and multiple reaction chambers set with primers. The liquid delivery unit comprises a drive source and a rotating shaft, and the heating unit comprises a heater and a lifting device. The imaging unit comprises a light source and a camera, and the display unit comprises a determination device and a display device.

Gene amplifications are an oncogenic driver utilized by many forms of cancer in tumor development or treatment relapse. Promoting genomic instability or the proclivity to propagate genomic alterations through acquired defects in DNA repair machinery, replication licensing, or cell cycle control, gene amplifications not only drive oncogenesis, but also afford an opportunity for therapeutic exploitation. Here, CRISPR-Cas9 nickases are disclosed which selectively promote cancer cell death in a gene amplification-dependent manner. For example, CRISPR- Cas9 nickases generate a lethal number of highly toxic single-ended double-strand breaks within the genome of proliferating cancer cells harboring amplified genomic loci during DNA replication. Cas9 nickases may serve as a tumor-selective therapeutic that mitigates the collateral damage observed with conventional chemoradiotherapies and avoids chemoresistance.

The invention provides a molecular diagnosis method for avian leukosis virus and Marek's disease virus mixed infection. The molecular diagnosis method comprises the following steps: (S01) collecting diseased material tissues; (S02) carrying out nucleic acid extraction on the sick material tissue of the sick chicken; (S03) detecting the nucleic acid by using specific primers of avian leukosis and Marek's disease; (S04) preparing a PCR (Polymerase Chain Reaction) detection system, and respectively adding ALV-env gene amplification primers and MDV-meq gene amplification primers for gene amplification; (S05) carrying out 1% agarose gel electrophoresis detection on the amplification product in the step (4), and carrying out sequence determination after determining a target band; (S06) carrying out BLAST comparison on the sequences obtained by sequencing, carrying out gene sequence analysis and drawing an evolutionary tree; and (S07) determining the virus type according to the homology and the evolutionary branches, so as to realize rapid diagnosis of ALV and MDV mixed infection, strain characteristic analysis and exclusion of other similar epidemic diseases, and provide technical support for accurate prevention and control of AL and MD in the poultry industry, provenance purification and epidemiological investigation.

To provide a method for efficiently and accurately distinguishing two similar diseases that differ in the presence or absence of a mutant allele in the same gene region.SOLUTION: A method for distinguishing between a genetic disease caused by a codon mutation in a gene and another disease includes the steps of: performing an amplification reaction to amplify a genetic region containing the codon, the amplification reaction including adding a DNA primer for amplifying the genetic region containing the codon and a PNA that inhibits the amplification of a normal allele containing the codon; and treating a product obtained by the amplification reaction with a restriction enzyme that specifically recognizes and cleaves either a base sequence containing the codon with the mutation or a base sequence containing the codon without the mutation, thereby obtaining a restriction enzyme-treated product.SELECTED DRAWING: Figure 3

The invention relates to the technical field of PCR instruments, in particular to an HLA-A gene amplification PCR instrument and an amplification method thereof.The HLA-A gene amplification PCR instrument comprises a PCR instrument body, and a supporting bottom ring is fixedly installed in the PCR instrument body; sample holes are formed in the microwell plate, and the microwell plate is covered with a plate cover; the plate body supporting assembly is movably mounted on the supporting bottom ring, and the microporous plate is supported through the plate body supporting assembly; the plate body fixing assembly is installed on the plate body supporting assembly, the microwell plate is fixed through the plate body fixing assembly, and the plate body fixing assembly further fixes the plate cover; the invention also relates to an HLA-A gene amplification method. The method comprises the following steps: 1, extracting DNA of a sample; 2, carrying out PCR (Polymerase Chain Reaction) amplification on the HLA-A gene; step 3, electrophoresis detection; 4, purifying and diluting a PCR product; step 5, sequencing amplification; 6, purifying a sequencing amplification product; and 7, sequencing on a machine.

The application discloses a gene amplification detection method based on nucleic acid mass spectrometry technology, comprising the following steps: setting a target gene region, configuring a reference gene, and confirming experimental primers based on the target gene region and the reference gene; obtaining an experimental sample, performing a primer reaction processing operation based on the experimental primers and the experimental sample, and obtaining a sample to be analyzed; setting an interpretation interval, configuring a sample applicator and a nucleic acid mass spectrometer, performing a data analysis and interpretation operation based on the sample to be analyzed, the interpretation interval, the sample applicator and the nucleic acid mass spectrometer, and obtaining a gene amplification interpretation result; the application can achieve a detection accuracy higher than that of the fluorescence in situ hybridization technology and the immunohistochemical technology in the prior art, a detection throughput higher than that of the fluorescent quantitative PCR technology and the digital PCR technology, a detection cost lower than that of the fluorescent quantitative PCR technology and the digital PCR technology, and a detection time shorter than that of the second-generation sequencing NGS technology.

The invention discloses a thermal cover assembly of a gene amplification instrument. The thermal cover assembly comprises a thermal cover bracket; the heat cover is mounted on the heat cover bracket; and the plurality of sealing elements are independently arranged on the hot cover and are used for realizing sealing in one-to-one correspondence with sample tubes of a porous plate placed on the gene amplification instrument body, and the sample tubes are sealed through the plurality of independent sealing elements, so that the sealing effect is good.

The application discloses a kind of binarization observation methods and systems of gene network expression state, it is related to the field of life science cell gene expression determination technical field, method includes: according to gene dictionary and the gene dimension of measured cell determines random measurement matrix, and then determines the multiple gene name groups corresponding to random measurement matrix;For each gene name group corresponding to the random measurement matrix, T7 is used to customize positive value primer and negative value primer respectively, and is labeled respectively, to obtain corresponding positive value labeled primer and negative value labeled primer;Using positive value labeled primer, the gene data corresponding to positive value gene name group is amplified, and the gene data corresponding to negative value gene name group is amplified using negative value labeled primer, to obtain comprehensive gene amplification data, and it is labeled to obtain gene observation data;According to gene observation data and gene dictionary, the gene expression of measured cell is calculated.The application improves gene expression measurement stability.

The invention relates to the technical field of gene amplification, in particular to a PCR gene amplification device which comprises a shell, an amplification groove is formed in the upper portion of the shell, a heat conduction plate is fixedly installed at the bottom of the amplification groove and penetrates to the lower portion of the amplification groove, a semiconductor Peltier is installed below the heat conduction plate in an attached mode, and a water cooling structure is arranged below the semiconductor Peltier. The water cooling structure comprises a water cooling box fixedly installed below the semiconductor Peltier and a heat dissipation piece fixedly installed on one side of the shell, a water inlet pipe and a water outlet pipe are arranged on the water cooling box, a storage piece is connected to the water outlet pipe in parallel, and the storage piece is used for conducting heat preservation storage on liquid flowing out of the water outlet pipe. According to the semiconductor Peltier element, a cold source or a heat source generated below the semiconductor Peltier element can be fully utilized, and the cold source or the heat source is not directly discharged.

The invention discloses a primer group, a kit and a method for identifying deer species based on IGF1 (Insulin-like Growth Factor 1), IGF2 (Insulin-like Growth Factor 2) and RAF1 genes. The primer group comprises three pairs of specific primers aiming at IGF1, IGF2 and RAF1 genes, and the sequences of the three pairs of specific primers are respectively SEQ ID NO: 12 and 13, SEQ ID NO: 14 and 15, and SEQ ID NO: 16 and 17. In specific identification, DNA of a sample is extracted, PCR amplification is carried out by using the primer group, and then the fragment length difference of an amplification product is analyzed through agarose gel electrophoresis. By comparing specific length combinations of IGF1, IGF2 and RAF1 gene amplified fragments, the four types of cervidae animals can be accurately distinguished. According to the application, the identification system for the cervidae species based on the IGF1, IGF2 and RAF1 genes is established for the first time, and the identification system has the advantages of rapidness, accuracy, low cost and no need of sequencing, and is suitable for species source identification and market supervision of related products.

A method for predicting responsiveness to a HER2-directed therapy by assessing HER2 heterogeneity in a tumor includes contacting a sample of the tumor with a biomarker-specific reagent that specifically binds to HER2 protein and detecting HER2 protein in the sample, contacting the sample of the tumor with a first nucleic acid probe that specifically binds HER2 genomic DNA and detecting HER2 gene amplification status in the sample, contacting the sample of the tumor with a second nucleic acid probe that specifically binds HER2 RNA and detecting HER2 RNA status in the sample scoring the HER2 protein (IHC), HER2 gene (DISH), and HER2 RNA (RNA-ISH), predicting that the tumor is responsive to the HER2-directed therapy if the tumor reveals a first foci having a first score and a second score, in which the first score and the second score are not the same.

The invention discloses a method for identifying aquilaria agallocha and aquilaria sinensis by using a KASII gene. The agilawood and the aquilaria sinensis are identified on the basis that the KASII genotype of the agilawood is TT and the KASII genotype of the aquilaria sinensis is AA. 20 # basic groups of KASII gene coding regions of aquilaria sinensis and aquilaria sinensis are taken as target sites, a PCR (Polymerase Chain Reaction) and Sanger sequencing method is adopted, a PCR amplification product of the KASII gene is subjected to Sanger sequencing, if the genotype of the KASII is TT, the product is the aquilaria sinensis, and if the genotype of the KASII is AA, the product is the aquilaria sinensis. According to the method for identifying the agilawood agilawood and the aquilaria sinensis through the KASII gene, high-throughput sequencing is carried out on the agilawood agilawood and the aquilaria sinensis, genotype detection analysis is carried out in a whole genome range, and it is found that the KASII gene has significant difference between the agilawood agilawood and the aquilaria sinensis, and the gene function is clear. The method is used for identifying the germplasm of the agilawood agilawood and the aquilaria sinensis by utilizing the genotype difference of the agilawood agilawood and the aquilaria sinensis and adopting PCR amplification and Sanger sequencing methods

The present disclosure provides a primer-probe combination, kit, method and application for detecting methylation of gastric cancer-related genes. The method comprises: collecting and separating venous blood to obtain plasma, extracting gene fragments of free RNF180 gene, SEPTIN9 gene, DACT2 gene, SDC2 gene and NKD2 gene in the plasma for standby use; converting the above gene fragments into sulfite, designing a fluorescent PCR reaction system to amplify the gene fragments; comparing and analyzing the CT value of the obtained gene amplification result with the critical value, when the CT value is less than or equal to the critical value, it is positive, indicating that the collected plasma contains at least one of the methylated genes. By adopting the primer-probe combination disclosed in the present disclosure, unnecessary large gastroscopy can be avoided when performing detection, and the whole process can be simply detected in vitro using peripheral blood, which is non-invasive, simple and easy.

The invention discloses a primer group of an HLA gene, a detection kit and a detection method, and belongs to the technical field of biomedical detection. The primer group of the HLA gene is composed of nine groups of primers, and the nucleotide sequences of the primers are shown as SEQ ID No.1-SEQ ID No.18 in a sequence table. The HLA genotyping detection kit comprises a primer group of an HLA gene; according to the HLA genotyping detection method, the primer group of the HLA gene or the HLA genotyping detection kit is adopted for detection. According to the method, 11 HLA genes are amplified by using a unified PCR condition, the coverage degree of amplicons on the HLA genes is high, the analysis resolution is high, and the method is suitable for large-scale and high-precision HLA typing detection.

The invention discloses a detection method for noninvasive prenatal detection of fetal trisomy 21 syndrome based on digital PCR, and relates to the technical field of digital PCR. According to the invention, a target area detection amplification primer and a Taqman-MGB probe are designed according to different sequences of human chromosome 21 genes, and a specific common amplification primer and a specific Taqman-MGB probe are designed according to similar sequences of the human chromosome 21 genes. Accurate detection of a low-proportion trisomy 21 syndrome positive sample is realized, and the omission ratio is greatly reduced. Compared with next-generation sequencing, multiple qPCR technologies and the like, the detection method provided by the invention has the advantages of simplicity in operation, simplicity in data analysis, short detection period, low cost and the like, meanwhile, by innovatively using a similar sequence design technology, high consistency of amplification efficiency of a detection gene and a reference gene is ensured, and the detection accuracy is improved. The detection sensitivity and accuracy are obviously improved.

The invention provides a detection method for detecting Sf9 host DNA residues by using a fluorescent probe and application, and belongs to the technical field of biological detection.According to the method, a specific primer pair (such as SEQIDNO: 1 / 2) and the fluorescent probe (such as SEQIDNO: 11) are designed according to a high-copy conserved sequence of Sf9 host DNA, a kit containing a gene amplification solution, a standard substance and a quality control substance is matched, DNA is extracted through a liquid nitrogen grinding-column extraction method, and the detection result is accurate. And carrying out real-time fluorescent quantitative PCR amplification and then carrying out detection. According to the method, the detection sensitivity reaches 0.003 pg / mu L, the linear range is 300 pg / mu L-0. 003 pg / mu L, cross reaction to irrelevant DNA is avoided, the repeatability CV% is smaller than or equal to 10%, the reagent is still stable after being frozen and thawed for 10 times, and the whole process is completed within 1.5 hours; the method solves the problems of insufficient specificity and sensitivity, tedious operation and the like of the existing method, and can be used for quality control of biological products such as gene therapy drugs and cell vaccines.

The purpose of the present invention is to establish a simple, efficient, and rapid genetic testing method for bacteria in clinical settings, and to provide: a gene amplification method for specifically detecting and evaluating the amount of bacteria associated with periodontal disease and caries; a primer set for a gene amplification reaction; a kit for detecting genes of bacteria that cause periodontal disease or caries, the kit including the primer set; and a kit for evaluating the severity of periodontal disease or caries. The present invention is a method for detecting oral bacteria that are present in plaque or saliva above or below the gingival margin, or bacteria that cause periodontal disease or caries, by detecting genes of the oral bacteria or causative bacteria using a gene amplification method, the detection method being characterized in that (1) annealing and extension reactions in the gene amplification method are implemented at substantially the same temperature, and / or (2) the gene length of a target gene as an amplification product is short.

This application relates to a molecular diagnostic reference for human gene detection, its preparation method, and its application. The reference is a recombinant cell, and the chromosome of the recombinant cell integrates the exogenous human variant gene to be detected in the form of homologous recombination; wherein the recombinant cell is selected from either recombinant mammalian cells or recombinant insect cells. This reference is substantially similar to clinical samples or cell lines of the human variant gene to be detected in terms of structure, performance, and composition, and can fully simulate gene deletion, gene insertion, gene fusion, gene amplification, fragment duplication, gene breakage, SNP, and gene overexpression. Furthermore, the preparation method of the reference is simple to operate, has a short preparation cycle, produces large quantities, has low production costs, and its source and performance are sufficiently stable. During use, the entire process from sample processing to detection can be monitored, exhibiting high biosafety and making it well-suited for application in molecular diagnostic products for human gene detection.

Disclosed are methods of genetic engineering to manipulate gene copy number in vivo, as well genetic constructs for amplifying gene copy number in vivo, and recombinant cells that comprise amplified genes. The methods of increasing gene copy number involve reducing expression levels of a haploinsufficient gene in the genome of recombinant cells, such as through replacing the endogenous promoter with a weaker promoter.