332 results about "Reference gene" patented technology

The invention belongs to the technical field of virus detection, and particularly relates to a primer, probe, test kit and method for real-time fluorescent quantitative PCR detection of novel coronavirus 2019-nCoV. A single-tube double-fluorescence channel is adopted to simultaneously detect the existence of the novel coronavirus 2019-nCoV and reference gene Rnase P, and the existence of the novelcoronavirus 2019-nCoV RNA in specimens such as alveolar lavage fluid, nasopharyngeal swab, whole blood, serum, feces and tissues can be detected. The method is short in detection time period, and issuitable for clinical and bedside rapid detection of diagnosis; the virus detection specificity is high, and the accuracy is high; virus qualitative analysis and quantitative analysis are carried out,and the quantitative linear range is good; the detection sensitivity is high; the experimental result is good in repeatability and high in precision; and the reference gene is added into a detectionsystem, so that the quality of the whole process of extracting and amplifying the sample can be monitored through the detection result of the reference gene.

The invention provides a kit for detecting 7 genetic markers of peripheral blood in early diagnosis of nasopharyngeal darcinoma. The kit comprises a peripheral blood total RNA extracting reagent, RT-PCR reaction liquid, time fluorescence quantitative RT-PCR reaction liquid and an SVM nasopharyngeal darcinoma diagnosis model, wherein the time fluorescence quantitative RT-PCR reaction liquid comprises primers of which the sequences are shown as SEQ IDNO:1-16; the primer sequences are SYBR Green fluorescence quantitative RT-PCR detection primers of the 7 genetic markers and reference gene GAPD; and the 7 genetic markers are HERC5, DLG7, PPARG, PLK1, KIF15, KLHL25 and MYST4. The invention also provides a using method for the kit. The kit has the advantages of high sensitivity, specificity and convenience, and can be used as aided diagnosis and early diagnosis technology for the nasopharyngeal darcinoma.

The invention belongs to the technical field of in-vitro nucleic acid detection and especially relates to a relative quantitative detection method of human motor neuron gene copy numbers and a kit thereof. According to the invention, specific primers and probes to the seventh and eighth exons of SMN1 and SMN2 genes and reference gene RPP40 are respectively designed; at the same time, qualitative detection primers and probes are also designed for three hot spot mutations of Y272C, 11bp-DUP and 4bp-DEL of the SMN1 gene. The kit comprises a container containing detection primer and probe compositions, a reference container and a PCR reaction liquid container. Relative quantitative determinations to the copy numbers of the seventh and eighth exons of SMN1 (in the first and second PCR reactions) and the seventh and eighth exons of SMN2 (in the third and fourth PCR reactions) and qualitative detection to the three hot spot mutations of Y272C, 11bp-DUP and 4bp-DEL (in the fifth PCR reactions) are respectively performed by 5 independent PCR reactions. The kit is strong in specificity, high in sensitivity, convenient, fast and applicable to large-scale popularization and application.

The invention discloses a kit for quantitatively detecting a BCR/ABL mRNA level. The kit comprises a standard product which is used for manufacturing a standard curve, an inner reference gene real-time quantitative PCR system and at least one of the following three real-time quantitative PCR systems: an M-type BCR/ABL real-time quantitative PCR system, m-type BCR/ABL real-time quantitative PCR system and a mu-type BCR/ABL real-time quantitative PCR system. The kit can accurately, quickly and quantitatively detect various BCR/ABL mRNA levels, is used for diagnosing chronic myelogenous leukemia and acute lymphoblastic leukemia expressed by BCR/ABL and monitoring minimal residual diseases in a treatment process, and provides an important molecular basis for accurate diagnosis of clinical diseases, determination of a treatment proposal, curative effect evaluation and prognosis.

The invention discloses a gene expression quantitative method and device. The gene expression quantitative method includes the steps that read section sequences containing nucleotide sequence information are obtained; the read section sequences are compared with all reference genes to obtain the read section sequences with successful comparison; the read section sequences with the successful comparison are filtered, the read section sequences with the soft shear ratio larger than a first preset value, the sequence length smaller than a second preset value and the comparison score smaller than a third preset value are rejected, and the ratio which the number of basic groups with unsuccessful comparison accounts for the total number of basic groups of each read section sequence serves as the soft shear ratio; the comparison score is a numerical value determined according to the matching degree of each read section sequence and the reference genes and the length of each read section sequence; for the filtered read section sequences, target gene expression is quantified by using RPKM. The read section sequences are compared with the reference genes instead of existing reference genomes, the comparison process can be simplified, and comparison efficiency can be improved.

The invention relates to a microRNA standardization reference gene and an application thereof. Specifically, with Solexa sequencing, real-time fluorescence quantitative PCR detection, literature screening, statistical analysis methods, the invention carries out researches upon a large amount of samples collected from a healthy control group and different disease patients. As a result, microRNAs let-7d, let-7g, let-7i, or combinations thereof can serve as microRNA standardization housekeeping gene. Compared with reference genes (such as U6, RNU44, RNU48 and miR-16, and the like) most commonly used in current microRNA quantification, the microRNA standardization reference gene has high stability and accuracy.

The invention discloses primers for detecting BCR/ABL fusion genes by a ddPCR technology and a detection method thereof. With digital PCR as a detection platform, in allusion to three types of BCR/ABLfusion genes, upstream and downstream primers for detecting the BCR/ABL fusion genes, a BCR/ABL fusion gene detection probe, upstream and downstream primers for detecting an internal reference gene and an internal reference gene detection probe are designed and synthesized, cDNA templates of a sample to be detected, the upstream and downstream primers for detecting the BCR/ABL fusion genes, the BCR/ABL fusion gene detection probe, the upstream and downstream primers for detecting an internal reference gene, the internal reference gene detection probe and a PCR premixed solution are mixed forpreparing ddPCR microreaction liquid drops for a PCR amplification reaction, and whether fusion gene templates are contained in the sample to be detected and the number and the content thereof are judged according to the types of fluorescence signals. The design specificity of the primers and the probes are strong, the detection sensitivity is higher through combination with the digital PCR technology, and a false negative result is avoided by automatically reading the result by software, so that the primers and the probes can be applied to detection of a small number of leukemia cells and thepossibility of relapse of a patient is reduced.

The invention provides a kit for detecting the copy number variation of an HER-2 gene by virtue of a digital PCR technique and a method. The kit comprises two groups of primer pairs and detection probes, a primer pair, a detection probe, a digital PCR premixed solution, luciferase, ddH2O and a calibration file, wherein the primer pairs and the detection probes are used for detecting the HER-2 gene, and the primer pair and the detection probe are used for detecting three reference genes CEP17, EIF2C1 and EFTUD2. Besides, the invention further provides a method for detecting the copy number variation of the HER-2 gene by virtue of the digital PCR technique. According to the method, phenomena of 17# chromosome multimer, aneuploid and centromere amplification can be eliminated, and the methodcan be used for accurately detecting whether the HER-2 gene is amplified to be positive or negative.

Disclosed are a data processing and analysis method of gene expression data for identifying endogenous reference genes and a composition for the quantitative analysis of gene expression, comprising a pair of primers and/or probes useful in the amplification of the identified endogenous reference genes. Introduced with the concepts of “Zero's proportion’ and CV, the me allows different datasets to be integrally analyzed, thereby searching for novel reference genes. By the method, 2,087 genes are first found as housekeeping genes which are expressed in most tissues, and the usefulness thereof in the relative quantification of different target genes is determined by analyzing their expression stability. Out of the 2,087 genes, 13 genes are found to show higher expression stability with lower expression levels across a wide range of samples than traditional reference genes such as GAPDH and ACTB, and therefore are suitable for the normalization of universal genes having relatively low expression levels.

The invention belongs to the technical field of analysis of gene expression and particularly relates to application of ClCAC gene and ClSAND gene as reference genes in analysis of gene expression of watermelon fruits. According to the invention, specific primers are designed for ClCAC gene and ClSAND gene. The specific primers cross over joint loci of two exons; no amplification product is generated when a genome DNA is used as a template; a target segment can be obtained through amplification when cDNA is used as a template; the nucleotide sequences of the primers are shown in SEQ ID No.1-4. Real-time fluorescence quantification PCR detection shows that both the ClCAC gene and the ClSAND gene can be expressed stably in different development stages of watermelon fruits which are different in gene type and fruit bearing mode and thus the ClCAC gene and the ClSAND gene can be used as a reliable reference gene combination in analysis of gene expression by real-time fluorescence quantification PCR detection. The reference gene combination has the advantages of good expression stability and the like and is not influenced no matter whether the genome DNA contamination exists in a cDNA sample or not.

The invention discloses a method for authenticating the copy number of target genes in a transgenic animal. The method provided by the invention comprises the following steps of: taking genome DNA of the transgenic animal as a template; performing fluorescence quantitative PCR by respectively using a target gene primer and a reference gene primer to respectively obtain a fluorescence quantitative PCR result of the target genes and reference genes of the transgenic animal; taking a standard substance with the target genes in a different copy number as the template; performing the fluorescence quantitative PCR by using the target gene primer and the reference gene primer respectively to obtain the fluorescence quantitative PCR result of the target genes and the reference genes in the standard substance; establishing a standard curve relative to the difference values of the cycle indexes of the target genes and the reference genes and the natural logarithm of the copy number of the target genes; and determining the copy number of the target genes in the transgenic animal. The method has the characteristics of simplicity, easy implementation, and accuracy and reliability because the results are consistent by using a conventional Southern blot method.

The invention provides a droplet-type digital PCR kit for COVID-19 nucleic acid test and an application of the kit. A reaction solution in the kit contains primers and a probe for detecting a COVID-19ORF1ab gene and a COVID-19 N gene, and primers and a probe for detecting human RNA reference genes, and two ends of each probe are labeled with fluorescent groups. The kit fully utilizes the characteristic of a small sample volume of microfluidic treatment, and realizes rapid heating and cooling of a sample, the maximum heating and cooling rate of the system can reach 10 DEG C/s, at the same time, the detection sensitivity to COVID-19 is 1 to 2 orders of magnitude higher than that of a traditional qPCR, and under the condition of a low virus concentration, the detection repeatability is better than that of the traditional qPCR.

The invention provides a kit for detection of animal origin components. The kit includes a membrane chip, the membrane chip is composed of a support membrane and probes fixed on the support membrane. The probes are specific probes for detection of pigs, sheep, yaks, donkeys and rats, the sequences of the probes are shown as Seq No.1-5, and the 5' end of each probe is labeled with amino. The kit also includes an internal reference probe for detection of internal reference gene, a positive probe and a negative probe, the sequences of the probes are shown as Seq No.6-8, and the 5' end of each probe is labeled with amino. The kit also includes positive oligonucleotide single stranded DNA with a biotin labeled 5' end, and the sequence is shown as Seq No.9. The kit provided by the invention can detect whether different varieties of meat products contain pigs, sheep, donkey, yak and rat 5 animal origin components substantially at a time, and the obtained detection result has the characteristics of strong specificity, high sensitivity, low false positive rate, high visual degree and low cost, etc.

The invention discloses a nucleic acid detection kit for Zika virus, dengue fever virus and Chikungunya virus, and an application of the kit, wherein the kit includes: a RT-PCR reaction solution, an enzyme mixture liquid, a Zika virus/dengue fever virus/Chikungunya virus/endogenous reference gene quadruple reaction solution, a positive control, and a blank control. The kit can simultaneously detect the Zika virus, dengue fever virus and Chikungunya virus in one reaction and solves a problem that an in-vitro detection kit cannot simultaneously detect the Zika virus, dengue fever virus and Chikungunya virus in one reaction tube; besides, the detection kit is high in specificity and sensitivity, has simple operation, excellent repeatability and quick and objection detection results. The invention provides an effective technical means for in-vitro detection of the Zika virus, dengue fever virus and Chikungunya virus.

The present invention provides a human immunodeficiency virus type 1 one-step fluorescence quantitative RT-PCR detection kit, which comprises a RT-PCR reaction solution, a RT-PCR enzyme mixture, a primer probe, a negative control, a strong positive control, a weak positive control, and calibration substances No.1-5. According to the present invention, a one-step fluorescence quantitative reaction can be directly performed on the extracted HIV-1RNA, the virus loading of the HIV-1RNA in a sample can be detected, the reference gene is adopted as the internal control, and the UNG enzyme is adopted to prevent pollution; and the kit has characteristics of simple one-step amplification method, short procedure, easy operation, pollution prevention, strong detection result specificity, high sensitivity, clear result and high result reliability, and can be used for detection of the virus loading of the human immunodeficiency virus type 1 (HIV-1) RNA in blood serum.

The embodiment of the invention discloses a gene sequence processing method for making a CPU to output information of a first gene sequence according to a comparison result after gene comparison is conducted on the first gene sequence completely matched with a reference gene sequence in an FPGA. According the embodiment, the method comprises the steps that a field-programmable gate array (FPGA) obtains gene sequences sent by the CPU; the FPGA selects rules according to preset gene sequences, and the first gene sequence in the gene sequences is determined; the FPGA conducts matching on the first gene sequence and the reference gene sequence through a preset algorithm; if the first gene sequence and the reference gene sequence are completely matched, the FPGA sends the matching result of thefirst gene sequence and the reference gene sequence to the CPU, and the matching result is used for determining output processing conducted by the CPU on the information of the first gene sequence.

The invention provides a multicolor fluorescence PCR kit and method for detecting HLA-B*5801 allele. The multicolor fluorescence PCR kit is characterized by comprising a detection primer-probe combination of two forward primers, two reverse primers and three detection probes for detecting the gene HLA-B*5801 and an internal-control primer probe comprising a forward primer, a reverse primer and an internal-control probe for detecting a reference gene. Based on the primer design and PCR reaction characteristics, the time and consumables can be saved to a great degree, the detection process only needs 1-1.5 hours, the operation is simple and easy to implement, and the whole experiment can be finished within 2.5 hours.

The invention discloses a novel multifunctional dual-luciferase reporter gene plasmid. The plasmid contains a luc firefly luciferase gene, a Rluc renilla luciferase gene, a SV40 poly(A) termination signal part and two independent multiple cloning sites, wherein the two independent multiple cloning sites are located on the upstream and the downstream of a luc firefly luciferase gene coder frame respectively. The dual-luciferase reporter gene plasmid containing detection gene and reference gene is successfully constructed, not only can the detection gene be transferred into a subject cell conveniently, but also the reference gene can be taken in correspondingly, the complexity of experiment operation and experiment errors are greatly reduced, the plasmid has the multiple cloning sites at thetwo ends of the firefly luciferase gene, the multiple cloning site located on the upstream of firefly luciferase can introduce a promoter sequence which can be used for genetic transcription adjustment, control and detection, and the multiple cloning site located on the downstream of the firefly luciferase can introduce a 3'UTR sequence which can be used for miRNA target identification, so that switching of different application of the same plasmid is achieved.

The invention discloses a reagent kit and a method for detecting human alpha defensin 1/3 gene copy number. The reagent kit comprises a first group of primers, i.e. target gene real-time fluorescence quantitative PCR (Polymerase Chain Reaction) amplification primers, a first fluorescent probe, i.e. a target gene real-time fluorescence quantitative PCR amplification fluorescent probe, a second group of primers, i.e. a reference gene real-time fluorescence quantitative PCR amplification primers, a second fluorescent probe, i.e. a target gene real-time fluorescence quantitative PCR amplification fluorescent probe, a calibration sample DNA (Deoxyribose Nucleic Acid), and real-time fluorescence quantitative PCR reaction liquid, wherein base sequences of the first group of primers is shown by SEQ (Sequence) ID (Identity) No. 1 and 2; a middle probe base sequence is shown by SEQ ID No. 3; base sequences of the second group of primers are shown by SEQ ID No. 4 and 5; a middle probe base sequence is shown by SEQ ID No. 6. The reagent kit for detecting the human alpha defensin 1/3 gene copy number has the advantages of simplicity, convenience and quickness in use, no standard curve required, high sensitivity and specificity, good repeatability and high detection efficiency and can be used for indicating the risk prediction of clinical infection immune diseases.

The invention discloses a special primer for testing the minRNA-128 in colorectal cancer serum. The special primer comprises a reverse transcription primer and a test primer of the miR-128, a reverse transcription primer and a test primer of U6 and a reverse transcription primer and a test primer of miRNA-16, as shown by SEQ IDNO: 1-9. A special kit for testing colorectal cancer serum minRNA-128 comprises the primers and a RNA separation solution. A method for testing the expression amount of the colorectal cancer serum minRNA-128 comprises steps: mixing a serum specimen to be tested with an isovolumetric RNA separation solution, centrifuging 16000g of the mixture for 10 minutes, and separating supernate; performing RT-PCR (reverse transcription-polymerase chain reaction); manufacturing a standard curve; and performing calculation to obtain a gene calibration initial copy number Q, and comparing the Q value of the miR-128 and the geometrical mean of Q values of reference gene U6 and miR-16 to obtain a relative expression amount of the minRNA-128. According to the method for testing the minRNA-128 in colorectal cancer serum, important references are provided for early discovery and early treatment of colorectal cancer.

The invention relates to an internal reference gene capable of stable expression in different tissues of Sogatella furcifera, and a screening method and application thereof, belonging to the technical field of biology. The nucleotide sequence of the internal reference gene RPL9 is disclosed as SEQ ID NO:7. The method comprises the following steps: designing degenerate primers according to other insect related gene sequences disclosed in NCBI, carrying out PCR (polymerase chain reaction) to obtain 6 common internal reference gene partial nucleotide sequences of Sogatella furcifera, and carrying out cloning, sequencing and NCBI database Blast comparison to determine the partial sequences of the Sogatella furcifera internal reference gene; and designing specific quantitative primers on the basis of the partial sequences, establishing an RT-qPCR (reverse transcription-quantitative polymerase chain reaction) process based on an SYBR Green I dye technique, and screening out the internal reference gene RPL9 capable of stable expression in different tissue parts of Sogatella furcifera by using a fluorescent quantitative PCR technique. The internal reference gene lays solid foundation for researching gene expression level of different tissue parts and gene functions of Sogatella furcifera in future.

The invention discloses a swine-derived component real-time fluorescent PCR (polymerase chain reaction) detection method, which comprises the steps of: designing swine specific primers, external primers and internal primers of an internal reference GCG-R gene; conducting PCR amplification on a gene fragment with a length of 270bp in the internal reference GCG-R gene; diluting the gene fragment by 10 times with DEPC (diethypyrocarbonate) water; extracting DNA of a sample; employing the swine specific primers, the internal primers and the diluted gene fragment to carry out swine-derived component real-time fluorescent PCR detection on the DNA. The invention also discloses the primers adopted by the swine-derived component real-time fluorescent PCR detection method. The primers include the swine specific primers, the external primers and the internal primers. By introducing an internal reference gene GCG gene template and its primers, the method can accurately, effectively, and rapidly detect swine-derived components, and the detection result is accurate and reliable. Compared with the current identification technologies, the method provided in the invention can realize accurate and fast identification that whether meat food contains a swine-derived component.

The invention discloses a plasmid reference molecule which is suitable for the quantitative determination of nucleic acids from a transgenic soybean GTS40-3-2, comprising a structure-specific sequence and a strain-specific sequence of the transgenic soybean GTS40-3-2 and a specific fragment of a soybean endogenous reference gene Lectin. The invention is characterized by designing primers to obtain the structure-specific sequence and the strain-specific sequence of the transgenic soybean GTS40-3-2 and the specific fragment of the soybean endogenous reference gene Lectin by PCR amplification through the analysis of inserting the exogenous source of the transgenic soybean GTS40-3-2 in the gene sequence; constructing by molecular cloning to form a artificial recombinant plasmid molecule pXL02 in a plasmid molecule; detecting the phosphorus content of the plasmid reference molecule by inductively coupled plasma mass spectrometry (ICP-MS) to calculate the concentration of the plasmid reference molecule. According to the invention, the plasmid reference molecule constructed in the invention can completely replace a transgenic soybean GTS40-3-2 positive standard sample, and the plasmid reference molecule is completely suitable for the quantitative PCR analysis and detection of structure specificity and strain specificity of the transgenic soybean GTS40-3-2 sample.

The invention relates to a reference gene for fluorescence quantification of different tissues of Chinese yam and primers and application thereof, and belongs to the technical field of molecular biology of Chinese yam. The nucleotide sequence of the reference gene CKI-2 is as shown in SEQ ID NO. 1. The nucleotide sequence of a PCR amplification primer of the reference gene CKI-2 is as follows: anupstream primer is shown as SEQ ID NO.2; and the downstream primer is as shown in SEQ ID NO. 3. Protein kinase I, myb, F-box, actin gene, EF1, bHLH13, eIF1, grid heavy chain protein and other genes are selected as candidate genes of different tissues. The stability of candidate genes is evaluated through qRT-PCR in combination with RefFind, reference genes stably expressed in different tissues ofthe Chinese yams are selected out through screening, and a reference basis is provided for later development of research work such as gene function verification of growth and development and metabolicmechanisms of the Chinese yams.