316 results about "SYBR Green I" patented technology

The invention aims to develop a flow-cytometry-based method for rapidly measuring heterotrophic bacteria in a eutrophic lake. The method is a flow-cytometry-based bacterium counting method. The method comprises the following steps of fixing a sample, performing ultrasonic processing, filtering, performing SYBR Green I dyeing, detecting the heterotrophic bacteria by utilizing a flow cytometer, and acquiring forward and lateral scattering light, an SYBR Green I green fluorescence signal and a chlorophyll a red fluorescence signal of autotrophic plankton, wherein the SYBR Green I green fluorescence signal is used for setting a threshold value; and eliminating the influence of the nannoplankton on bacterium detection by utilizing the forward and lateral scattering light, separating the plankton from abiological particles and cellular debris according to the intensity of the SYBR Green I green fluorescence signal, and separating the bacteria from phytoplankton according to the intensity of the chlorophyll a red fluorescence signal of the autotrophic plankton, thereby obtaining the accurate number of the heterotrophic bacteria. The method has the time-saving and labor-saving effects, the large-scale ecological survey can be developed, and the counting precision and accuracy of the bacteria are improved.

The invention discloses a fluorescence detection method of cysteine. In the method, mercury ion specific DNA (mismatching of 7 pieces of thymine-thymine) is in a random curled state without the presence of mercury ions; after the mercury ions are added, a thymine-Hg2<+>-thymine (T-Hg2<+>-T) complex is formed, so that the mercury ion specific DNA has a hairpin-shaped structure; fluorescent dye Sybr Green I, which can be combined with double-chain DNA to emit light and has high fluorescence intensity, is added; and amino acids to be tested are added, when the cysteine is detected, the cysteine and the mercury ions can form a more stable complex, so that the mercury ions can be extracted from the T-Hg2<+>-T complex, the hairpin-shaped structure unwinds, the fluorescence intensity is reduced, and other amino acids cannot cause the change of the fluorescence intensity. The method is high in sensitivity and specificity, simple and quick; and the whole process can be finished within 5 minutes.

The invention discloses a ginseng identification method and a special kit. The method comprises the steps: a) extracting genomes DNA as templates from a to-be-tested sample and utilizing primers (sequence 1 and sequence 2) to perform PCR (Polymerase Chain Reaction) amplification; b) adding a fluorescent dye SYBR Green I to the amplified reaction system and determining whether the to-be-tested sample contains ginsengs according to the following methods; if green fluorescence is observed in the reaction system, the to-be-tested sample or the candidate product contains ginsengs; if no green fluorescence is observed in the reaction system, the to-be-tested sample or the candidate product does not contain ginsengs. The experimental results show that the rapid and accurate identification of ginsengs, American ginsengs, pseudo-ginsengs, panax japonicus, rhizoma panacis majoris and bipinnatifid ginseng rhizome can be realized by the method provided by the invention; the fluorescent dye method is introduced to detect true or false identification results and technical supports are provided to realize onsite application of identification of medicinal molecules.

The invention relates to a method for detecting miRNA (micro Ribose Nucleic Acid) by an improved stem-lop primer qRT-PCR (Quantitative Reverse Transcription Polymerase Chain Reaction). The method comprises the steps: (1), designing a miRNA detection primer, wherein a stem-loop primer comprises two parts of a general stem loop sequence and a specific primer sequence which is in complementary pairing with a 3' end of a target miRNA, a base number which is in complementary pairing with the target miRNA is 11bp, a downstream primer needle of qPCR is specific to a general stem loop sequence, an upstream primer needle is specific to a 5' end of the target miRNA, a GC tail is added in the 5' end of the upstream primer so that the annealing temperatures of the upstream primer and the downstream primer are close; (2), performing inverse transcription on the target miRNA into cDNA (complementary Desoxvribose Nucleic Acid); and (3), performing a quantitative PCR amplification and detecting by adopting an SYBR Green qPCR detection method. The method disclosed by the invention is good in specificity, and high in sensitivity; and one downstream primer is only synthesized, and an SYBR Green I fluorochrome method is adopted, and thus the cost is greatly saved.

The invention discloses a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay kit and a detection method for grass carp reoviruses (GCRV), and belongs to the technical field of the detection of fish viruses. The RT-LAMP assay kit for the grass carp reoviruses comprises the following ingredients: 1*ThermoPol Reaction Buffer, Bacillus stearothermophilus deoxyribonucleic acid (BstDNA) polymerase, deoxyribonucleotide triphosphate (dNTPs), AMV reverse transcriptase, outer primers (F3 and B3), inner primers (FIP and BIP), Betaine, MgCl2, dithiothreitol (DTT) and 1,000*SYBR Green I. The assay kit has the characteristics of simpleness, convenience, quickness and high specificity and sensitivity; GCRV-104 in a sample can be accurately detected within 2 hours only by using a water bath kettle or metal bath; and the assay kit can detect grass carp tissues infected by the GCRV-104 and cells (such as cytokine-induced killer (CIK) cells) infected by the GCRV-104, and is applicable to the on-site rapid detection of the GCRV-104.

The invention discloses a specific primer for assaying genetically modified corn MIR162, the sequence of which is shown as SEQ ID NO:1 and SEQ ID NO:2. The invention also discloses a fluorescently labeled probe for assaying genetically modified corn MIR162, the sequence of which is shown as SEQ ID NO:3, the 5' end of the probe is connected with a fluorescent reporter FAM, and the 3' end of the probe is connected with a fluorescent quencher Eclipse. The invention also discloses an assay method which utilizes the primer and the probe to carry out gene-specific qualitative PCR (Polymerase Chain Reaction), SYBR Green I real-time fluorogenic quantitative PCR and TaqMan probe real-time fluorogenic quantitative PCR to assay whether corn and related products thereof contain MIR162 transformation events, and an experiment proves that the assay method is sensitive, accurate, simple and reliable, and has a high application value and a wide market prospect.

The invention discloses an LAMP detection kit for Koi herpesvirus and a detection method. The LAMP detection kit comprises a 10*ThermoPol Reaction buffer liquor, Bst DNA (Deoxyribose Nucleic Acid) polymerase, dNTPs, outer primers F3 and B3, inner primers FIP and BIP, Betaine, MgCL2, and 1000*SYBR Green I. The LAMP detection kit provided by the invention has the characteristics of simplicity, convenience, rapidness and high specificity and sensitivity, and KHV (Koi Herpesvirus) in a sample can be accurately detected within 2 hours by a water bath kettle or a metal bath. The diseased fish tissue infected by KHV can be detected, and cells infected by KHV can be detected, so that the kit is very suitable for field fast detection of KHV.

The invention discloses a haemophilus parasuis real-time fluorescent quantitative PCR (polymerase chain reaction) detection method which selects the 16S rRNA gene (AB004028) which has high conservation and specificity and is the main target gene of bacteria classification and determination as the amplification target gene, takes a high-conservation area of the gene as an amplification area to design a specificity primer, and establishes a haemophilus parasuis real-time fluorescent quantitative PCR detection method embedding a fluorescent dye SYBR Green I and DNA, wherein the DNA of the initial template of the haemophilus parasuis 16S rRNA gene can be accurately quantified according to the standard curve of the known standard product, and equated to the number of bacteria. The method disclosed by the invention has strong generality and high specificity, can detect many HPS serotypes, is free from influence of related bacteria, and can be applied to quick and accurate quantitative detection of the haemophilus parasuis seriously impairing development of the pig industry.

The invention provides a SYBR Green I detection method for detecting HLA-B*1502 gene and its special primers and kit. The primers are designed according to the 2nd and 3rd exon regions of the HLA-B*1502 gene, and are used for qualitative detection of the HLA-B*1502 gene in the sample to be tested. The upstream primer of the primers has the nucleotide sequence of SEQ ID NO: 1 in the sequence listing, and the downstream primer has the nucleotide sequence of SEQ ID NO: 2 in the sequence listing. The invention can quickly and accurately detect the HLA-B*1502 gene, and is of great significance for ensuring human health and the drug safety of carbamazepine drugs. The detection method and the kit of the present invention can be used for nucleic acid detection of the HLA-B*1502 gene in various clinical samples (bone marrow, peripheral blood or tissue), and have broad application prospects.

The invention discloses a dual SYBR Green I real-time fluorescence polymerase chain reaction (PCR) detection primer and a dual SYBR Green I real-time fluorescence PCR detection method for porcine pseudorabies virus and porcine circovirus type 2. The primer is designed and synthesized, and the dual SYBR Green I real-time fluorescence PCR detection method for the porcine pseudorabies virus and the porcine circovirus type 2 by using the primer comprises the following steps of: extracting deoxyribose nucleic acid (DNA) of a sample; and detecting the sample by using an SYBR Green I real-time fluorescence PCR reaction system and an SYBR Green I real-time fluorescence PCR amplification program. By the primer and the method, two viruses, namely the porcine pseudorabies virus and the porcine circovirus type 2 can be detected simultaneously, and porcine parvovirus, classical swine fever virus, porcine reproductive and respiratory syndrome virus and swine influenza virus cannot be detected. The primer and the method have the characteristics of higher sensitivity, repeatability and stability, and contribute to the identification and the diagnosis of pregnant sow reproductive disturbance virus disease.

The invention discloses cucumber phytophthora LAMP (Loop-mediated Isothermal Amplification) primer and a rapid detection method thereof, which are specifically used for the specific detection of cucumber phytophthora. The rapid detection method is characterized in that a cucumber phytophthora LAMP primer is mainly adopted and designed, detailed information can be seen in SEQ NO.1-SEQ NO.4. Green fluorescent light or an LAMP characterized ladder-shaped pattern can be observed through the LAMP and the color developing by adding an SYBR green I color agent for developing color or the agarose gel electrophoresis detection. The LAMP primer and the rapid detection method, which are disclosed by the invention, can be used for quickly, sensitively and accurately detecting the cucumber phytophthora in plants and soil which are affected by the cucumber phytophthora in production practice and can be simultaneously used for the early diagnosis of diseases in filed and the monitoring and the identification of germs, and reliable technical and theoretical basis is provided for preventing the disease caused by the cucumber phytophthora.

The invention belongs to the field of biotechnology, particularly relates to a titer detection method for a slow virus carrier, and discloses a quality index detecting method conducting real-time fluorescence quantification nucleic acid amplification detection (qPCR and RT-qPCR) by using a sequence specific primer and a fluorescent dye, a used primer and a standard product. The detecting method isefficient and accurate, the operation is easy, the use amount of a sample is low, the repeatability is good, and the limit problem of inaccurate carrier quantitation in the fields of eucaryon geneticengineering and gene therapy is solved. The method has the advantages of detection time and simple and convenient operation step, the analysis process after the amplification is not needed, comparedwith the Taqman probe method, the SYBR Green I does not need the design of a synthesis fluorescent probe, the design is simplified, the cost is lowered, and a dissolution curve is combined in the analysis to judge the specificity of the reaction.

The invention discloses a method for rapidly detecting pig source components in red meat through a real-time fluorescence loop-mediated isothermal amplification (LAMP) method. According to the method, the pig specific cytochrome b genes in the red meat are detected by employing a method for combining the LAMP technology with real-time fluorescence. According to the detection method, the genome DNA of the meat to be detected is extracted, six specific primers and Bst DNA polymerase fragments with strand displacement activity are amplified at the temperature of 60-65 DEG C for 45-60 minutes, SYBR GREEN I is added into the reaction system, and the amplification conditions of the sample template can be detected in real time, so that whether pig source components are contained in beef and mutton samples can be judged. The method has the advantages of high sensitivity, high specificity, simplicity and rapidness, and the result is not required to be subjected to gel electrophoresis.

The invention relates to a realtime fluorescent quantitative PCR detection method of cyanobacteria producing microcystin (MC) for rapidly, accurately and quantitatively detecting cyanobacteria producing microcystin (MC) in eutrophic water body of a freshwater lake. The method comprises the following steps of: collecting and processing a lake water sample and extracting the total DNA; carrying out realtime fluorescent quantitative PCR detection of mcyA-CD gene fragment by using SYBR Green I chimeric fluorescence method; recording the fluorescence values and C(t) values of a standard curve sample, a positive reference, a negative reference and a sample to be detected; drawing a standard curve by a computer; and calculating the copy number of the mcyA-Cd gene fragments in the sample to be detected and the concentration of cyanobacteria producing MC. The inventive method can rapidly and accurately detect concentration of cyanobacteria producing MC in eutrophic water body of freshwater lake and has good repeatability.

The invention discloses an RT-LAMP (Reverse Transcription Loop-Mediated Isothermal Amplification) kit for detecting avian influenza virus subtype H7N9. The kit comprises six pairs of primers, the nucleotide sequences are shown in SEQ ID No.1-12 respectively. The kit can rapidly, specifically and sensitively detect the avian influenza virus subtype H7N9, a turbidity meter is utilized for judging the reaction in real time, or SYBR Green I is injected after the reaction is finished, and a detection result is judged by color reaction; the lowest detection limit of a primer for detecting HA gene is 48fg/mul of H7N9 RNA; and the lowest detection limit of a primer for detecting NA gene is 4.8pg/mul of H7N9 RNA. The kit is convenient and simple to use, low in cost and good in specificity, is very suitable for disease surveillance, on-site emergency and clinical specimen detection, and is suitable for large-range popularization and application, and the reaction result is easy to observe.

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 kit for detecting the proviral DNA (Deoxyribonucleic Acid) of an HIV (Human Immunodeficiency Virus). An isothermal amplification reagent is placed in the kit and contains an aqueous solution of primers LTR-F3, LTR-B3, LTR-FIP and LTR-BIP, an aqueous solution of MgCl2, an aqueous solution of dNTP, a 10*betaine buffer, a 20*SYBR dye, a BstDNA polymerase and 3.9 microliters of sterile water for injection. According to the kit, after a loop-mediated isothermal amplification reaction system is successfully synthesized, the system is combined with a fluorescent dye, the SYBR Green I fluorescent dye is combined with amplified fragments along with the amplification, the fluorescence intensity is recorded by a fluorescent quantitative PCR (Polymerase Chain Reaction) instrument, and finally, the virus content is given according to the fluorescence intensity.

An ARMS-based method for detecting botryis cinerea SdhB gene H272Y mutation belongs to a real-time fluorescent quantitative detection method of gene mutation, integrates the real-time PCR technology and the ARMS technology, and greatly reduces the interference of a primer dimmer on the detection process through special primer design, so as to improve the detection sensitivity and accuracy. Moreover, the SYBR Green I dye method is adopted to reduce the detection cost.

The invention discloses a kit for detecting riemerella anatipestifer and a method for using the kit. At least two pairs of specific primers F3, B3, FIP and BIP for loop-mediated isothermal amplification are placed in the kit for detecting riemerella anatipestifer. For making use convenient, the kit also comprises lycine, magnesium sulfate, dNTPs, ThermoPol reaction buffer and Bst DNA polymerase; or a color developing reagent SYBR GREEN I is also added. The kit and the method have the advantages of convenient application on base layer, short detection time, low cost and simple and convenient operation.

The invention provides a real-time fluorescence quantitative PCR detection method of grass carp reovirus SYBR Green I, at least comprising the following steps of: extracting the total RNA of the grass carp reovirus, designing a primer, carrying out RT-PCR (Reverse Transcription-Polymerase Chain Reaction) amplification on the coat protein gene of the grass carp reovirus, cloning and identifying the coat protein gene of the grass carp reovirus, diluting a standard template and establishing a fluorescence quantitative PCR standard curve. The detection method has low requirements on the concentration of the template and high detection sensitivity and can carry out quantitative analysis. The detection method can detect 72 samples at a time; a result can be observed immediately once the PCR reaction is finished without the operations of electrophoresis, dyeing and the like, and therefore, the detection method is more convenient and rapid than conventional RT-PCR methods.

The invention provides a method for detecting bovine viral diarrhea viruses as well as a preparation method and a use method of a reverse transcription loop-mediated isothermal amplification (RT-LAMP) reaction kit, relating to a preparation method and a use method of a reaction kit. The invention solves the problems that the traditional method for detecting bovine viral diarrhea viruses has expensive cost, needs professional equipment and professional staff to participate in detection, and is not suitable for the development state of the animal husbandry at the present stage of China. The preparation method comprises the following steps: firstly, designing a primer, preparing RT-LAMP reaction liquid, and then, finishing the preparation. The use method comprises the following steps: adding RNA extract containing bovine viral diarrhea virus samples into the reaction liquid, ungergoing RT-LAMP reaction after mixing, and then, centrifugally detecting; or adding 10,000*SYBR GREEN I, and putting below an ultraviolet lamp for detecting; or carrying out electrophoresis detection; or carrying out insulation reaction detection by adopting a Real-time PCR instrument. The reaction kit has convenient detection, does not need expensive instruments and reduces the cost.

The invention discloses a general nucleic acid isothermal amplification detection kit for pathogenic vibrios of mariculture animals. The detection kit comprises a grinding liquid tube into which grinding liquid is filled, a nucleic acid extracting solution tube A into which solution of sodium acetate is filled, a nucleic acid extracting solution tube B into which absolute ethanol is filled, a nucleic acid extracting solution tube C into which 70 mass percent ethanol solution is filled, a tris-hydrogen chloride ethylene diamine tetraacetic acid (TE) buffer solution tube into which TE buffer solution is filled, a uracil-DNA-glycosylase (UNG) tube into which uracil-DNA-glycosylase is filled, a loop-mediated isothermal amplification (LAMP) reaction liquid tube into which LAMP reaction liquid is filled, a bacillus stearothermophilus deoxyribonucleic acid (Bst DNA) polymerase tube into which Bst DNA polymerase is filled, a color-developing agent tube into which a nucleic acid dye SYBR Green I is filled, a positive control nucleic acid tube into which positive DNA of the vibrios is filled and a negative control tube into which sterilized double distilled water is filled. The invention also discloses a method for detecting the pathogenic vibrios of the mariculture animals by utilizing the detection kit.

The invention provides a method for detecting feline herpes virus. The method comprises the following steps: screening to obtain an oligonucleotide primer, and detecting feline herpes virus by adopting a SYBR Green I fluorescent quantitative PCR method. The invention provides a primer pair for detecting feline herpes virus, and the sequences of an upstream primer and a downstream primer are respectively SEQ ID NO:1 and SEQ ID NO:2. The screened primer not only can ensure the accuracy of identified pathogen, but also can not detect congener different strains of pathogen. In addition, the amplified segments have a proper length, thus not only being capable of separating from each other in electrophoresis, but also being incapable of dispersing, and avoiding the situation that small segments are optimally amplified and the amplification of long segments are blocked in multiple PCR; and non-specific amplified products such as a primer dimer and the like. in addition, the high-specificity and high-sensitivity fluorescent quantitative PCR detection method for FHV can be obtained by the optimization of a fluorescent quantitative reaction system and reaction conditions.