161 results about "Molecular epidemiology" patented technology

The invention discloses a multiple PCR method for simultaneously detecting and identifying three human tissue echinococcosis pathogens, including echinococcus granulosus G1-G3, echinococcus multilocularis and echinococcus granulosus G6-G10. According to the invention, 3 pairs of specific primers are designed for the mitochondrial genes of 3 pathogens; the specific primers are added into a same PCR reaction system; the specific primers are complementarily combined with the target genes of the corresponding parasite species; through amplified reaction, different parasite species can generate target stripes in different sizes; gel electrophoresis is adopted for separating and detecting. The three pairs of primers according to the invention are free from mutual interference and have no cross reaction with 8 tapeworms in close genetic relationship or do not interfere with the result judgment. The method is high in specificity and sensitivity, can effectively and accurately realize the synchronous detection for 3 pathogens, can greatly save the detection time and cost, can effectively increase the working efficiency and can be applied to the research on the parting identification for human echinococcosis pathogens and echinococcosis molecular epidemiology.

The invention belongs to the biotechnology application field, and relates to simultaneous detection and genotyping of infections of 16 respiratory viruses (including FluA, FluB, sH1N1, PIV1, PIV2, PIV3, RSVA, RSVB, HRV, HMPV, HBoV, CoV NL63, CoV OC43, CoV 229E, CoV HKU1 and Adv) of nasopharyngeal extract specimens of patients of respiratory-related diseases of all levels of disease prevention and control institutions and sentinel hospitals. Specifically, nucleotide sequences of representative strains of the 16 respiratory viruses are downloaded from NCBI; through literature review and multiple sequence alignment, pathogen relatively-conservative regions are determined, and multiplex specific primers are designed. Single-tube multiplex (18 stages) PCR detection is carried out for detecting the 16 respiratory virus conservative regions, and an entire reaction takes less than 2 hours. According to the invention, a defect that genotyping cannot be carried out with conventional single-tube multiplex fluorescent qualitative PCR detections can be overcome, and defects of complicated operations, long time, and high cost of conventional chip detection methods can be overcome. With the application provided by the invention, a novel idea is provided for respiratory virus genotyping technologies. With characteristics of high specificity, high sensitivity, and high speed, powerful technological support is provided for rapid and accurate screening and genotyping of the respiratory-disease-related viruses. The invention has important significance upon the researches of respiratory-tract patient infection pathogen spectrum of out nation, and upon molecular epidemiological investigations.

The invention belongs to the field of biomedical detection, relates to the technical field of microbe molecule biology detection, in particular to a multiplex polymerase chain reaction (PCR) kit capable of quickly identifying and diagnosing mycobacterium tuberculosis, nontuberculous mycobacteria, mycobacterium bovis and Bacillus Calmette Guerin vaccine simultaneously, and discloses four primer pair sequences designed for specific segments of four differential genes, namely 16S rRNA, IS6110, Rv2652 and Rv3873 of the mycobacterium, and amplification conditions for multiplex PCR. By the invention, a mycobacteria tuberculosis complex sequence can be quickly and accurately amplified in a sputum sample of a tuberculosis patient, and species identification can be carried out; the sensitivity is 50 colony forming units/template, which is far higher than that of an acid-fast staining method, an inspection procedure for the mycobacterium tuberculosis is greatly simplified, and a detection speed is improved; and meanwhile, different mycobacterium strains can be identified through once PCR, and a technical means is provided for clinic quick diagnosis of tuberculosis and molecular epidemiology survey.

The invention discloses a multiple PCR detection kit for virulence factors of streptococcus suis and a detection method thereof, and belongs to the technical field of biology. The multiple PCR detection kit comprises nucleic acid shown by base sequences, such as SEQ ID NO:1 to SEQ ID NO:20. The detection method for the multiple PCR detection kit comprises the following steps of: 1, providing DNA of samples to be detected; 2, taking the kit, amplifying the DNA of the samples to be detected by adopting the conventional PCR method, detecting an amplified result by adopting an agarose gel electrophoresis method, and judging according to the result; and by using positive DNA of streptococcus suis 2 type as contrast, if the contrast proves that not all the strips are amplified, re-detecting, and if the contrast proves that all the strips are amplified, judging the result. The multiple PCR detection method established by the invention is specific and sensitive, and has the advantages of simpleness, convenience and quickness. The multiple PCR detection kit has reliable stability, and can be used for rapid detection of clinical samples and survey research on molecular epidemiology for the streptococcus suis.

The invention belongs to the technical field of molecular biology, and particularly relates to a real-time fluorescent quantitative PCR primer, kit and detection method for detecting the porcine circovirus 3. The nucleotide sequence of the real-time fluorescent quantitative PCR primer for detecting the porcine circovirus 3 is shown in SEQ ID NO.1-2. The invention further provides the kit containing the primer mentioned above and the detection method for detecting the porcine circovirus 3. The primer, the kit and the detection method are high in specificity, high in sensitivity and stable in repeatability, wave crests of the melting curve of amplified products are single, no primer dimer is caused, and the primer and the kit have no cross reaction with genomes of the porcine circovirus 2, the hog cholera virus, the porcine pseudorabies virus and the porcine reproductive and respiratory syndrome virus; sensitivity is 102 copies per microliter and is 100 times higher than that of the conventional PCR; and the variation coefficients of inter-group and intra-group repeated tests are all smaller than 3.0%. The foundation is laid for research on molecular epidemiology, prevention and control of the pestilence.

The invention provides a kit for detecting cysticercus cellulosae, swine trichinella and swine toxoplasmosis. The kit comprises sterile deionized water, polymerase chain reaction (PCR) liquid, thermus aquaticus deoxyribonucleic acid (Taq DNA) polymerase, a GoldView DNA dye, bromophenol blue sample loading buffer solution, a standard substance and a reference substance. In the detection kit provided by the invention, DNA of the cysticercus cellulosae, the swine trichinella and the swine toxoplasmosis in a specimen to be detected can be detected quickly and accurately only by one reaction, and the kit can also be used for the molecular epidemiological survey and curative effect monitoring of cysticercus cellulosae, swine trichinella and swine toxoplasmosis infection. The method has simple preparation steps and is low in cost, easy to operate, time-saving and labor-saving, a large number of reagents and consumables are saved, and the work efficiency is improved. The method is high in detection sensibility, specificity and accuracy, and the false positive rate is reduced.

The invention provides a method for detecting avian paramyxoviruses, which detects RT-PCR of avian paramyxoviruses (APMV) by using specific primers. The method comprises the following steps of: performing extraction and reverse transcription of total sample RNA to obtain sample cDNA; amplifying target fragments by using the specific primers; performing gel electrophoretic analysis; and judging results. A pair of specific detection primers is designed according to conservative region sequence of NP genes of different serotype avian paramyxoviruses, the sequence is as shown in Seq ID NO:1 and Seq ID No:2, and target gene fragments of 387bp are amplified. The method has the characteristics of high specificity, high flexibility, high efficiency, low cost, suitability for quickly detecting the different serotype avian paramyxoviruses as well as analyzing and detecting a large number of samples and extremely important significance for quickly diagnosing the avian paramyxoviruses at early stage as well as analyzing molecular epidemiology, and simultaneously provides technical means for research on the molecular variation mechanism of the avian paramyxoviruses.

The invention discloses a method for detecting hantaan virus strain genome. The method has the following steps: (1) two pairs of hantaan virus general primers are designed; (2) the hantaan virus detection method comprises the following steps that: first, viral specific RNA extraction kit is utilized to extract the total RNA in a serum sample; second, a nucleic acid analyzer is adopted to detect the RNA content, and the RNA is taken to perform the RT-PCR reaction; third, RT-PCR:RT uses HV group-specific primers such as P0, PCR uses HV general primers such as P1P2 and P3P4; fourth, viral nucleic acid amplified results are observed through agarose gel electrophoresis, and a specific nucleic acid band appears. The method is intuitionistic and predominant, the sensibility is good, and the effects are good; the serum detection rate in recent ten years is 79.2 percent, and the serum detection rate in twenty years ago can still reach to 70.5 percent; the method is applied to the laboratory detection of hantaan virus infection and the molecular epidemiological survey.

The invention provides a Fusobacterium nucleatum subsp. animalis strain THCT6b3 separated from human intestines. A 16SrRNA gene sequence of the strain is shown in SEQ ID NO:1. The classification nameof the strain is Fusobacterium nucleatum subsp. animalis and collected on May 17, 2019 with the collection number of CCTCC NO:M 2019363 in the China Center for Type Culture Collection. The invention further provides application of the strain in detection of Fusobacterium nucleatum. A PCR-based molecular identification method for Fusobacterium nucleatum subsp. animalis is established for the firsttime. The strain can promote disclosing of composition and sources of the Fusobacterium nucleatum in intestines of colorectal cancer patients, and contributes to analysis of molecular epidemiologic characteristics of the Fusobacterium nucleatum.

The invention discloses a multiple PCR detection primer of enterococcus, comprising a sequence of the enterococcus primer, a sequence of enterococcus faecalis strain primer and a sequence of enterococcus faecium strain. The invention establishes a multiple PCR method for identifying the enterococcus fast and accurately, greatly simplifies the checking program, and can be used in a number of basic laboratories. The method can identify the enterococcus and faecalis or faecium by one-time PCR, can detect 1ng genomic DNA at least, has high sensitivity and stronger specificity, is beneficial to identification and diagnosis of the enterococcus, has better sensitivity, repeatability and stability. The invention provides a new fast detection method, and also provides test basis and technical means for molecular epidemiology survey of enterococcal infection, study of enterococcus molecule pathogenic mechanism and early diagnosis, research and development of a diagnostic reagent kit and immunologic mechanism of drugs or vaccines.

The invention relates to a Cyprinid herpesvirus detection kit and a detection method thereof, belonging to the technical field of virus genome detection. The detection kit comprises a 10* reaction buffer solution, a 1U/mu L Taq enzyme, a primer set composed of 10 mu M of P1F and 10 mu M of P1R, a primer set composed of 10 mu M of P2F and 10 mu M of P2R, pure water and a 15 mu g/mL positive DNA. The kit can be used for simultaneously detecting genomes of carp pox herpesvirus, haematopoietic necrosis herpesvirus of goldfish and Koi herpesvirus. The method has the advantages of excellent visuality, favorable sensitivity and high specificity, has favorable effects on detecting trace amounts of Cyprinid herpesvirus genome in the long-storage-time specimen, and can be used for Cyprinid-herpesvirus-infected laboratory detection and Cyprinid herpesvirus molecule epidemiological survey.

The invention provides a special prime designed according to conservative M genes of influenza virus. By the aid of the principle of specific binding of biotin and streptavidin, biotin-labeled PCR (polymerase chain reaction) products are bound on a microtiter plate, anti-digoxin peroxidase is added, and finally TMB (tetramethylbenzidine) color development solution is added, so that positive products can develop colors. On the basis, a special kit is successfully prepared. The method combines PCR with a high-sensitivity digoxin detection system, the sensitivity of the method is 100 times higher than that of a conventional agarose gel electrophoresis detection method, and the method is high in specificity and overcomes difficulty in detecting AIV (avian influenza virus) of a low content in a tissue sample. Compared with a virus isolation method, the method has the advantages that coincidence rate can reach more than 95%, pollution of chemical agents (such as ethidium bromide) is eliminated, automation is benefited, the method is suitable for detecting a large number of samples, and a new approach is provided for early diagnosis of avian influenza and molecular epidemiological investigation.

The invention relates to a novel saliva preserving fluid comprising the following components: 1-10mmol/L of EDTA, 1-50mmol/L of Tris-HCl, 10-100 mmol/L of sodium chloride; 0.1-3% (w/v) of sodium sorbate, 0.1-3% (w/v) of sodium diacetate, 0.01-0.1% (v/v) of proclin 300, 0.1-1% (v/v) of TWEEN20, 0.1-1% (w/v) of NP40 and 0.1-2% (w/v) of SDS. The novel saliva preserving fluid is used for preservingsaliva samples, can effectively inhibit nuclease activity, prevent microbial pollution, guarantee DNA fragment integrity and purity, simplify pretreatment steps, is suitable for centrifuge-free and low-temperature environments, can guarantee transport stability of samples among different places, and is suitable for molecular epidemiological large-scale population investigation and research.

The invention discloses a rapid detection kit for goat contagious pleuropneumonia and a preparation method thereof. The kit is established on the basis of molecular biology. The kit comprises 2*loop-mediated isothermal amplification (LAMP) reaction liquid, a primer mixer, BstDNA polymerase, a color-developing agent, a standard positive template and ddH2O, wherein the primer mixture comprises a forward inner primer FIP, a downward inner primer BIP, a forward outer primer F3 and a downward outer primer B3; and the ratio of the FIP to the BIP to the F3 to the B3 is 8:8:1:1. The kit has the characteristics of high sensitivity and specificity and the like, can react quickly, is easy to operate, does not need special instrument equipment and overcomes the defects of long pathogeny separation time, high workload, fussy detection method, complicated operation and the like of goat contagious pleuropneumonia, and the detection method is complicated, is difficult to operate and the like, and can be used for detecting whether clinical samples are infected with Mycoplasma capricolum subsp. Capripneumonia as well as performing early diagnosis and molecular epidemiological survey of goat contagious pleuropneumonia.

The invention discloses a whole genome sequence of Seneca valley virus SVV/CH/ZZ/2016 and an amplification primer thereof. The whole genome sequence of Seneca valley virus SVV/CH/ZZ/2016 is obtained by the following steps: amplifying 7 nucleotide sequence fragments (S1, S2, S3, S4, S5, S6 and S7) of the strain of SVV/CH/ZZ/2016 by a one-step RT-PCR method; performing clone sequencing on the 7 nucleotide sequence fragments; sequentially splicing, editing and correcting the DNA sequences of the 7 nucleotide sequence fragments, to finally obtain the whole genome sequence of Seneca valley virus SVV/CH/ZZ/2016. The whole genome sequence of Seneca valley virus SVV/CH/ZZ/2016 obtained in the invention is favorable for further studying the pathogenic mechanism, molecular epidemiology, reverse genetics and the like of the Seneca valley virus, and provides important data support and theoretical basis for the diagnostic reagent development, vaccine development and the like of the Seneca valley virus.

The invention discloses a molecule detection method for puccinia striiformis f.sp.tritici No.17 physiological race. By employing a method for converting a RAPD molecular marker into a SCAR molecular marker, the specific primers (upstream primer 5' -TTCCCCCGCTGTCT and downstream primer 5'- GTTGAGCGGGGGAA) of No.17 physiological race are obtained, and are subjected to PCR amplified reaction (reaction conditions: 94 DEG C, 5 min, 1 cycle; 94 DEG C, 30 s, 60 DEG C, 1 min, 72 DEG C, 90 s, 35 cycles; 72 DEG C, 7 min, 1 cycle) and 2% agarose gel electrophoresis, so that an specific strip with a length of about 1000 bp is obtained. Therefore, the molecule detection method helps to rapidly discriminate and distinguish puccinia striiformis f.sp.tritici No.17 physiological race in molecular level, and establishes a base for developing research on wheat stripe rust molecular epidemiology.

The invention discloses a genotype inspection method and a genotyping inspection method of staphylococcal enterotoxin. The genotype inspection method of staphylococcal enterotoxin includes the steps of sample treatment, DNA extraction, PCR and PCR amplified product analysis. The amplified sample is put in agarose gel including ethidium bromide for electrophoresis and color development and observed under a transmissive uviol lamp; the inspected sample can be judged as a positive sample including corresponding staphylococcal enterotoxin genotype if the length of the amplified nucleic acid band is the same as the amplified length in table 2. The first method can fast inspect the particular staphylococcal enterotoxin genotype, with high sensitivity, high particularity and high accuracy. The second method can achieve systematic genotyping of staphylococcal enterotoxin genes from different sources and provide technical support for analysis of relationships among different enterotoxin types. The invention is significant in food security quarantine, disease diagnosis and molecular epidemiology research.

The invention provides a multiplex PCR detection primer and method for important viruses causing egg-laying abnormality. The method comprises the steps of preparing specificity capture probes aiming at viral nucleic acids of three important viruses such as an egg-laying descending syndrome virus, an avian Tembusu virus and an H9 subtype avian influenza virus which cause the egg-laying abnormality, carrying out hybridization on the specificity capture probes and the corresponding viral nucleic acids, then carrying out cyclization in the presence of ligase, carrying out PCR amplification by virtue of a pair of universal detection primers specific to the probes so as to detect, and determining infection pathogeny according to the size of an obtained amplified product. The method can be used for detecting a single virus and simultaneously differentially diagnosing three different viruses, has the characteristics of high efficiency, specificity and sensitivity and low cost, is applicable to the detection analysis of a large number of clinical samples when laying fowl cannot normally lay eggs, and is an important technological measure for the early rapid differential diagnosis and the molecular epidemiological analysis of pathogeny which definitely causes the egg-laying abnormality.

The invention provides a reagent kit for detecting avian leukemia virus J sub-groups (ALV-J), and belongs to the technical field of RT-PCR (reverse transcription-polymerase chain reaction) detection. The reagent kit contains a pair of specific primers. Nucleotide sequences of the specific primers are respectively shown as SEQ ID NO.1-2. The invention further provides a method for detecting the avian leukemia virus J sub-groups. The reagent kit and the method have the advantages of high specificity, sensitivity and efficiency, good universality and low cost. Besides, quick differential diagnosis can be carried out on clinical disease samples in 6.5 h, technical means can be provided for early quick diagnosis on the ALV-J and conduction of molecular epidemiological investigation, and accordingly avian leukemia prevention and control can be effectively guided during poultry raising production.

The invention provides the method checking the pig adding red cell body PCR, the used reagent includes the PCR checking reagent box, the positive comparison containing the aiming gene segment recombining matter grain, the negative comparison, the Tap enzyme, the GoldView DNA dye, the cushioning liquid, the testing goal of the checked sample can be reached using the DNA distilling, PCR enlarging and adding and enlarging and adding outcome. The method provided by the invention can test the adding red cell body of the checked sample quickly and truly, so it can be used to the survey of the molecule epidemiology of the pig adding red cell body illness. The templet DNA has many merits such as the easy handling, the low cost, removing the jam of the bacilli and the impurity during the checking by the lens, improving the diagnosing nicety greatly and depressing the percentage of the fake masculine.

The invention relates to STR (short tandem repeats) molecular markers for Sporothrix globosa and applications of the STR molecular markers. The invention provides a microsatellite locus (STR molecular marker) combination for the Sporothrix globosa for the first time and provides a set of specific primers (SEQ ID NO:26-75) capable of efficiently amplifying Sporothrix globosa populations as well as detection conditions. The microsatellite locus combination contains 25 highly polymorphic microsatellite loci (SEQ ID NO:1-25). The set of microsatellite loci for the Sporothrix globosa not only can be applied to genetic background analysis and molecular tracing of the Sporothrix globosa, but also can be applied to molecular epidemiology, population genetics and phylogenetic analysis of the Sporothrix globosa.

The invention relates to a method for analyzing molecular typing based on gastric mucosal lesion proteomics, different gastric mucosal lesion proteomics molecular subtype characteristics and correlation between the molecular typing and the subtype characteristics and the gastric mucosal lesion progress. A protein marker database related to gastric cancer and gastric mucosal lesion progress is established by calculating the relationship between protein expression and gastric mucosal tissue pathological state, proteome molecular subtype and gastric mucosal lesion progress, and a gastric mucosallesion sample disease progression risk scoring system is established. According to the invention, a molecular epidemiological research means is combined with bioinformatics analysis and machine learning, microscopic and macroscopic gastric cancer cause risk factors are integrated, a gastric mucosal lesion molecular typing framework and a progress risk prediction model are established, and a foundation is laid for finally constructing a comprehensive and systematic gastric cancer prevention strategy.

The invention provides a kit for detecting NDV (Newcastle disease viruses) of chicken with different genotypes, and belongs to the technical field of RT-PCR (reverse transcription-polymerase chain reaction) detection. The kit comprises a pair of specific primers with nucleotide sequences respectively as shown in SEQ ID NO. 1-2. The invention further provides a method for detecting the Newcastle disease viruses of the chicken with different genotypes. The kit and the detection method have the advantages of high specificity, sensitivity, efficiency and universality and low cost, clinical samples can be rapidly differentiated and diagnosed within 6 hours, and technical means are provided for early and rapid diagnosis of the NDV and molecular epidemiological investigation, so that the NDV can be more effectively prevented and controlled in poultry raising production.

The invention discloses a whole genome sequence of a rabies street virus HN10, shown in SEQ ID No. 1. The invention also provides an amplimer of the whole genome sequence, shown in SEQ ID No.2 and SEQ ID No. 49. In the invention, the virus genome sequence is divided into 24 segments, corresponding cDNA is amplified by PCR method, a fragment primer is used for direct sequencing, the used primer is designed based on a nucleotide sequence in the conservative area of rabies viruses, the end of a primer 3' is terminated in the position of little amino acid degenerating codons, and a last basic group is terminated at a second-place basic group of an amino-acid codon. In the invention, the target fragment is effectively amplified, a whole genome sequence is provided. Therefore, the sequence is favorable for understanding the classification, evolution and pathogenicity of the virus and molecular epidemiology, etc. The virus is possibly used as the vector of a reverse genetic system, and the determination of the virus sequence lays a foundation for the construction of the virus vector.

The invention provides a reverse transcription-polymerase chain reaction (RT-PCR) method for verifying avian infectious bronchitis virus (IBV) epidemic strains and vaccine strains by multi-primer combination. The method comprises the following steps of sample total ribose nucleic acid (RNA) extraction, sample complementary deoxyribose nucleic acid (cDNA) obtained through reverse transcription, target segment amplification through multi-primers and gel electrophoresis and result judgment. The method is applicable to the fast verification detection of ordinary vaccine strains and main epidemic wild strains of avian IBV, and has the characteristics of high specificity, high sensitivity, high efficiency and low cost, the verification of IBV and other viruses is realized, and meanwhile, the attribution of strain genotype is verified. The establishment of the method has very important significance on both molecular variation mechanisms of the avian IBV and molecular epidemiology analysis.