CN105392896A - Rapid and sensitive genotype identification and nucleic acid detection - Google Patents
Rapid and sensitive genotype identification and nucleic acid detection Download PDFInfo
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- CN105392896A CN105392896A CN201480027795.3A CN201480027795A CN105392896A CN 105392896 A CN105392896 A CN 105392896A CN 201480027795 A CN201480027795 A CN 201480027795A CN 105392896 A CN105392896 A CN 105392896A
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Abstract
The invention discloses a method, primer, probe and kit for identifying various gene mutations or substance genetic typing. In one embodiment, the disease is pathogenic. In the other embodiment, the invention is applied to detection of multidrug-resistant Mycobacterium tuberculosis, hepatitis B virus, beta-globulin mutation, thrombophilia related mutation; or various sexually transmitted pathogens, consisting of Trichomonas vaginalis, Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma urealyticum, Ureaplasma parvum, Treponema pallidum, Herpes simplex virus 1 , Herpes simplex virus 2, Human papillomavirus type 6, and Human papillomavirus type 1.
Description
Related application
This application claims with the U.S. Provisional Patent Application numbers 61/791,933 submitted on March 15th, 2013 for basis for priority, all the elements of described patent application are all included in the application with way of reference.
Technical field
The field that the present invention relates to is to identify the various genotype relevant with human diseases.
Background technology
The present invention relates to identification different genotype.Sequence specific primers-polymerase chain reaction (SSP-PCR), DNA sequencing, DNA fingerprint, with technology such as single nucleotide polymorphism (SNP) gene types, be applied to gene type (PCT application publication number WO/2011/139750).In these techniques, single nucleotide polymorphism (SNP) gene type has higher insight.But the genotype tests that great majority relate to DNA hybridization all requires higher running cost and longer operating time, is therefore necessary to develop more effective methods of genotyping, reach to sooner, more cheap and cover more polygene type.
mycobacterium tuberculosis (MTB) detects
Tuberculosis (TB) is caused by mycobacterium tuberculosis (MycobacteriumtuberculosisMTB).The appearance of resistance in Mycobacterium tuberculosis (DR-MTB) is serious problems in industrialized country and developing country's Tuberculosis.Except the importance on clinical and epidemiology, drug-resistant tuberculosis (DR-MTB) more has important economic impact, because its treatment cost be height than common MTB.DR-MTB is defined as there is resistance at least two First Line medicines the most effective, comprises Rifampin (rifampinRIF) and vazadrine (isoniazidINH).DR-MTB case has the trend of rising in the whole world, the M & M shown.The resistance of one line antitubercular agent has been found relevant with several MTB transgenation: rpoB (resistance to RIF); KatG gene and inhA gene (resistance to INH).
The detection of DR-MTB has several different technologies to use.Although traditional phenotypic drug sensitivity test (DST) remains " the Yellow gold standard " of test MTB resistance, DST reaches possibly and just can complete in eight weeks.Mycobacterium tuberculosis is poky bacterium.Within the every 15-20 of MTB hour, just divide once, and depend on that used medium generally needed for 4 to 6 weeks just can grow into bacterium colony.After this, the MTB cultivated as DST is identified, and drug sensitive test also takes 2 weeks usually again.Therefore DST needs the long period, result in the delay of disease therapy, and especially common way wants begin treatment before DST completes.Alternative cultivation in industry exploitation maybe can shorten the time needed for test; But the MTB in some type of sample is not suitable for cultivating with substituting.Also infrequently use conventional DNA sequencing to detect MTB medicament-resistant mutation in commercial environment, because its consuming time and that needs are special knowledge.Market there is the test kit of different technologies, comprising PCR in real time and the conventional hybridization for detecting single type MTB or resistance MTB.Particularly PCR in real time, because its number of channels available is limited, so be usually difficult to detect several drug resistance MTB simultaneously.Although conventional hybridization is also applicable to detect multidrug resistance MTB; But it needs long incubative time (at least 4 hours).Under contrast, the present invention is by utilizing polymerase chain reaction (PCR) and " water conservancy diversion " hybridization technique, provide a kind of array test, can detect single resistance MTB and multidrug resistant MTB on a platform simultaneously, detection time is than conventional hybridization short (~ 35 minutes).Only need be less than 4 hours from extracting DNA sample to the whole test analyzed, detected result has specificity and the susceptibility of height.
the detection of beta Thalassemia
Beta Thalassemia is suddenlyd change by euchromosome and causes, and is a kind of hemoglobin disease, can classifies according to the affected degree of the production of betaglobulin again.Wherein some sudden change causes beta Globulin (to be expressed as β
+) the slight underproduction, some causes the absolute silence of betaglobulin gene and can not produce or only produce the beta Globulin losing function and (be expressed as β
0).According to estimates, the world about has the population of 1.5% to have the genotypic heterozygote of β-thalassemia, and father and mother are its offsprings of Words of heterozygote may be homozygote.Beta Thalassemia has three severity: slight (minor thalassemia, β
+/ β or β
0/ β), moderate (thalassemia intermedia: β
+/ β or β
0/ β) and serious (major thalaseemia: β
+/ β
+, β
0/ β
+or β
0/ β
0).The thalassemic people of heredity mainly there will be hypochrosis microcytic anemia, and may need lifelong blood transfusion.Control β-thalassemic best method avoids the birth of beta Thalassemia infant, and this wants accurate father and mother to do antenatal detection and genetic counseling; This has been proved to be and has the most effectively managed thalassemic method.β-thalassemia is popular in Temperate Region in China, as Mediterranean country, and the Middle East and south east asia.β-thalassemic molecular basis is existing to be widely studied, and finds that the point mutation of betaglobulin gene has different mutational formats and mutation frequency in different areas.
Current detection technique comprises microarray, ApoE gene (AS-PCR) and direct Sequencing.Microarray can detect on multiple target dna chip simultaneously, is therefore applicable to detect multiple sample simultaneously.But its application is the restriction because of its high cost, and result can not detect by an unaided eye, high-performance scanning device and complicated analysis software need be used to make an explanation.Is to use an allele specific allele specific PCR primers, to detect polymorphism or mutation, this technology only completely match with the target DNA oligonucleotides can act as primers for amplification of target DNA.The restriction of this technology is the limited throughput capacity a reaction.Direct DNA sequencing relate to external DNA replication dna process wherein archaeal dna polymerase optionally mix chain termination dideoxy nucleotide, the throughput capacity of this technology is low and need long-time operation, and therefore detection efficiency is low.On the other hand, the invention provides Yi Zhong Knot and close the DNA detection using PCR and reverse dot blot hybridization, the sudden change of the betaglobulin gene of an extremely sensitive and special detection can be provided, to assist β-thalassemic detection by Enough.
hepatitis B virus detects
Hepatitis B virus is minimum DNA virus, it comprise 3000 by Protein capsid around Nucleotide.Hepatitis B virus is by propagating with the blood or bioresorbable of sufferer.About 2,000,000,000 people infect hepatitis B virus; Wherein become hepatitis B virus carriers more than 3.5 hundred million.About 1/5th hepatitis b virus infected meetings develop liver cirrhosis or liver cancer.Detect hepatitis B virus and can pass through simple surface antigen blood testing, or use the measurement of using polymerase chain reaction (PCR) and hepatitis B virus DNA to diagnose.In addition, the difference by analyzing HBV genomic sequence has identified 10 kinds of genotype of hepatitis B virus (A to J) and several hypotype.Genotype of hepatitis B virus and hypotype obviously show different areal distribution.Various genotype of hepatitis B virus is causing a disease and treatment there are differences.The determination of genotype of hepatitis B virus can provide information for the assessment before chronic HBV infection and treatment.DNA sequencing is considered to detect Yellow gold standard method to genotype of hepatitis B virus.But it is lower in the genotypic efficiency of detection mixing.In addition, molecular dna technology provides one for extremely sensitive the concrete grammar detecting genotype of hepatitis B virus.
sexual reverse detects
Venereal disease is common worldwide disease.Estimate there are 448 Wan Xinfa every year and recoverable VC (syphilis, gonorrhoea, chlamydozoan and trichomonad), mainly occur in the grownup of 15 to 49 years old all over the world according to the World Health Organization in 2005.In 2010, every 550 pregnant woman had one to be detected suffering from sexual spreading disease (not comprising acquired immune deficiency syndrome (AIDS)) in Hong Kong.Venereal disease can infect the child of pregnant woman, no matter is at gestation time or after baby's birth.In the middle of, gonococcus and chlamydozoan can cause sterile.Therefore, accurate wedding Mr. and Mrs and pregnant front women have the potential demand detecting venereal disease.In addition, China is also the potential market detecting sexual reverse, and 20 years std patient quantity sharply increases in the past.Therefore China needs fast, and at a low price, sexual reverse check reagent box helps to control venereal disease accurately.Venereal disease check reagent box in the market can be used for detecting single pathogenic agent Huo Tong Time and detects 3-4 pathogenic agent, does not generally comprise Human papilloma virus HPV (HPV).Because the sickness rate of venereal disease increases, have and need screening strength and the multiple sexual reverse of detection simultaneously.
thrombophilia detects
Thrombophilia refers to that blood has abnormal solidifying, and adds and occurs thrombotic risk.Research report shows, and have in the thrombotic patient of thrombophilia Bing Chu Now, about 40% With inherited genetic factors is relevant, and has been proved high risk and suffers from the dysgenesias such as cardiovascular disorder and recurrent abortion such as venous thrombosis.Also have increasing suggestion to think to be hereditary the pregnant woman of thrombophilia more easily to occur pregnancy badness, symptom comprises recurrent abortion, Intrauterine Fetal Death, fetal intrauterine growth retardation, aura eclampsia and placental abruption etc.
Er Shi Inter in the past, some transgenations have been determined relevant with heredity thrombophilia.Modal four transgenations are: Leiden accelerator factor FactorVLeiden (FVL) [1691G>A], thrombogen FactorII (FII or be called factor II) [20210G>A], Methylene tetrahydrofolate reductase MethylenetetrahydrofolateReductase (MTHFR) [677C>T] and Methylene tetrahydrofolate reductase MethylenetetrahydrofolateReductase (MTHFR) [1298A>C].Detect these transgenations and contribute to identifying the high risk population having thrombophilia and recurrent abortion, thus help Ge Do personage's preventing disease and reduce risk, and preventative-therapeutic guidance is provided.
Brief summary of the invention
sNP gene type is as diagnostic tool
SNP gene type except can be used for DNA fingerprint, can also for the identification of gene fragment, Genetic polymorphism, there is the gene of change or the problematic gene of function.The invention provides method and test kit for fast, identify the communicable pathogenic agent of tool clearly, or the heredopathia caused by the existence of specific dna sequence or disappearance.
As above institute opinion, the hybridization format on market needs to be analyzed by the image analyzer of tool high solution degree of elephant.SNP gene type can be applied to equipotential specific oligonucleotide probe-reverse dot blot hybridization (ASO-RDB) the flow hybridization form (such as U.S. Patent number 5,741,647) on film As basis.Microarray hybridization form can produce visible point, and can be analyzed by the image analyzer of estimating and/or use cost is lower.
flow hybridization
DNA hybridization is modern biotechnology scientific research always and relates to the most important method in gene molecule biological study.Various genomic nucleotide sequence complicacy is hybridized revealed in the solution by the DNA sequence dna of particular complementary and analyzes.In brief, target dna is amplified or digests, and makes it hybridize with the complementary DNA probe on solid support matrix (as nitrocellulose filter).But traditional hybridization technique is limited to the area of film in principle, and usually need longer incubative time.
Flow hybridization method and apparatus can control hybridization conditions exactly, does not have conventional hybridization technology and takes and longer ask Questions.Hybridization time can be reduced to several minutes (whole hybridization assays can complete in 5-30 minute, and the concrete time depends on the method for generation of detection signal) from a few hours or a couple of days by water conservancy diversion DNA hybridization technology.Flow hybridization equipment cheap for manufacturing cost, and the amount of reagent using fewer than conventional hybridization equipment 10 times, therefore make DNA detection technology than front more economical material benefit.Flow hybridization technology provides sensitiveer, detect accurately and qualification result, and is generally applicable to the hybridization of various different technologies such as traditional southern blotting technique method (Southernblotting), RNA blotting (Northernblotting), dot blotting, slit blotting and reverse dot blotting.
PCT application WO/2011/139750 describes the multiple horizontal fast guiding test set that is connected to central control unit.Hybridization device comprises the central control unit that is connected to one or more lateral flow device.Lateral flow device carries out crossover process and developing programs, and is controlled by central control unit and power.Single lateral flow device or several equipment can test some reactions (or some samples and/or analyte) simultaneously, and carry out program with different condition under independently controlling.Lateral flow device can be the form of " nxm " dot matrix (matrix), also can be the form of linear array.Have the description of method and apparatus about performing flow hybridization can see United States Patent (USP) 5,741 by Ginseng more, and 647, United States Patent (USP) 6,020,187 and PCT application WO/2011/139750.Those skilled in the art can use United States Patent (USP) 5, and 741,647 or United States Patent (USP) 6,020, the nearly flow hybridization technology of the class described in 187, or any novel method of flow hybridization technology or equipment can be performed to put into practice the present invention.The imaging system that hybridization device has, such as FT-Pro, can be biometrics by signal detection.Hybridization device of the present invention can be the equipment of automatization, and the biometrics function that it can be used built-in is to carry out all analyses.Flow hybridization method has higher efficiency, accuracy and sensitivity than conventional hybridization technology.
drug resistant M mycobacterium (MTB) detects
The invention provides method and test kit and detect with PCR and " water conservancy diversion " formula hybridization technique the Mycobacterium tuberculosis (MTB) Rifampin (RIF) and vazadrine (INH) being had to resistance.The rpoB transgenation of the RNA polymerase β subunit of MTB is relevant with to the resistance of Rifampin; And katG gene (catalase) is relevant with to the resistance of vazadrine with the sudden change of INHA gene (enoyl-ACP reductase).In the present invention, Auele Specific Primer is used to amplification INHA, rpoB gene, and katG gene; Auele Specific Primer has been proved does not have cross reaction with human genome.Primer RS-IAC serves as internal control, and it is not for any mankind or MTB genome, for monitoring pcr amplification process.Probe includes ten for detecting the gene mutation DNA probe of resistance; This transgenation has rpoB (D516V, D516G, H526D, H526Y, H526L1, S531L and S531W), katG (S315T1 and S315T2), and inhA (-15TC/T).Probe also includes five for detecting the genomic DNA probe of wild-type MTB, and this contributes to checking rpoB gene, and whether the pcr amplification reaction of katG gene and inhA gene completes.Hybridized primer can be supported by biotinylation.
beta Thalassemia detects
The invention provides the method and test kit that detect betaglobulin transgenation, sudden change comprises TATA-28 (A>G), the detection of TATA-29 (A>G), initiator codon (G>A), codon 5 (-CT), codon 8/9 (+G), codon 15 (G>A), codon 16n (-C), codon 17 (A>T), codon 19 (A>G), codon 26 (G>A) (oxyphorase
), codon 27/28 (+C), codon is 30G>C, IVS1.1 (G>T), IVS1.1 (G>A), IVS1.5 (G>C), codon 41/42 (-TCTT), codon 43 (G>T), codon 71/72 (+A), the disappearance of IVS2.1 (G>A), IVS2.654 (C>T) and 619bp.
hepatitis B virus detects
The invention provides method and whether test kit is present in the serum sample of patient to detect 8 genotype of hepatitis B virus (genotype of hepatitis B virus A, B, C, D, E, F, G and H).
detect sexual reverse
The invention provides method and test kit, to detect in each sample, as urine, urogenital system swab (urethra, vagina, uterine cervix and various pathology) and liquid based cytology sample (PreservCyt
tMand SurePath
tM) protista, bacterium, and the existence of virus.Amplification controls (AC) and comprises for detecting human DNA, has enough cell contents and validity with check result.Whether the DNA that AC can be used to the existence and extraction of monitoring PCR inhibitor in pcr amplification process measures enough.There is no AC signal indication in the inhibitor of pcr amplification process or DNA quantity not sufficient (or there is not a corpse or other object for laboratory examination and chemical testing).Positive regulation (PC) comprises a positive template nucleic acid molecule to monitor the performance of PCR reagent.
DNA hybridization and round pcr can detect faster at urine, urogenital system swab (urethra, vagina, uterine cervix and various pathology), and the sexual reverse in liquid based cytology sample (PreservCytTM and SurePathTM).In one embodiment, described method and the detectable 12 kinds of common venereal diseases pathogenic agent of test kit: a kind of protozoon (Trichomonas vaginalis), 7 kinds of bacterium (chlamydia trachomatis, Diplococcus gonorrhoeae, mycoplasma genitalium, mycoplasma hominis, Ureaplasma urealyticum, solution sporozoite and treponema pallidum) and 4 kinds of viruses (herpes simplex virus 1 & 2 type, human papillomavirus 6 type and 11 types).These pathogenic agent are all relevant with trachelitis, urethritis, trichomoniasis and pelvic inflammatory disease.
Universal primer provided by the invention and pathogen specific primer, for detecting sexual reverse.Universal primer is used for realizing the PCR balance amplification simultaneously of multiple different target.Universal primer sequence tests specificity by PCR and gel electrophoresis, to prove there is not cross reaction with other pathogenic agent and human genome.Two universal primer sequence are added into the 5' end of pathogen specific primer and AC Auele Specific Primer respectively.Universal primer and pathogen specific primer mark are tested further by PCR and gel electrophoresis again, prove do not have cross reaction (Fig. 1) with other pathogenic agent and human genome.In one embodiment, pathogen specific primer and two universal sequences mark (primer) are included in single pcr amplification.At first round PCR, pathogen specific primer in conjunction with its specific target, and creates the pcr amplification product that is carried universal sequence.Then, take turns in PCR second, universal primer is attached to the PCR primer (Fig. 2) of universal sequence mark.
Under the PCR optimized, we can realize balance amplification and cause result to have deviation to avoid because different target pathogenic agent DNA sequence dna has different amplification efficiencies.Described embodiment also guarantees that the amplified production of pathogenic agent is only produced by universal primer, has detect more accurately with DNA probe.Universal primer system described herein also may be used on additive method and test kit detects the DR-MTB do not described in the present invention, beta Globulin, hepatitis B virus and thrombophilia, and the nucleic acid relevant with Other diseases or gene.
thrombophilia detects
The invention provides the method detecting the transgenation relevant with heredity thrombophilia, described transgenation comprises Leiden accelerator factor FactorVLeiden (FVL) [1691G>A], thrombogen FactorII (FII or be called factor II) [20210G>A], Methylene tetrahydrofolate reductase MethylenetetrahydrofolateReductase (MTHFR) [677C>T] and methyl tetrahydrofolate reductase enzyme MethylenetetrahydrofolateReductase (MTHFR) [1298A>C].
Although verification msg experiment of the present invention uses PCR with extension increasing sequence, the raw enough deals of any Ke Yi Productivity specific order Standard sequence also can be used with the method for carrying out qualification and flow hybridization analysis.If the order Standard sequence deal obtained is enough, just may not need to use pcr amplification.Detection can be realized by the mark detected on order Standard DNA or joiner.
Accompanying drawing explanation
Fig. 1 a shows the electrophoretic image using universal primer (sequence number 285 and 286) amplification clinical sample PCR reactant.Only in the positive (namely CT-is positive, and NG-is positive, HPV-6 and HPV-11 is positive) having pathogenic agent, observe special frequency band, this shows universal primer, and Hui and human DNA do not respond.Fig. 1 b shows the electrophoretic image of the PCR reactant using pathogen specific primer (sequence number 263-282) and universal primer (sequence number 285 and 286) amplifying human clinical sample.Each sample observation, to the special frequency band of corresponding special pathogen, shows that the pathogenic agent that primer is corresponding with it has high degree of specificity.Similar situation can be observed in single and multiple PCR reaction.
Fig. 2 shows the embodiment that is used in universal primer in single pcr amplification reaction and gene-specific primer.
Signal location (right figure) (ruling is way for instructions only) that the DR-MTB that Fig. 3 shows a DR-MTB case (left figure) and an embodiment detects.The amplification procedure of IAC internal control monitoring PCR.HC is that hybridization controls, monitoring crossover process.
Fig. 4 shows an example of the visual explanation of different MTB mutant.
Fig. 5 shows a kind of β-thalassemia case and signal location embodiment (ruling is way for instructions only).
Fig. 6 a shows one and explains the different genotypic examples of β-thalassemia.Fig. 6 b shows the test result of the various clinical samples using β-thalassemia case.
Fig. 7 shows the embodiment that hepatitis B virus test kit detects, and has the existence of 8 genotype of hepatitis B virus (A of hepatitis B virus, B, C, D, E, F, G and H).In universal primer IAC and HC is also included within.
Fig. 8 shows an example and goes to explain different genotype of hepatitis B virus.
Fig. 9 a shows the comparison of the susceptibility of the present invention and other manufactory's venereal disease test kits.The clinical sample of the venereal disease test kit of the performance of the present invention shown in Fig. 9 b and other manufacturer compares.
Figure 10 a shows a kind of venereal disease reagent array, the embodiment of case form and signal location.Described chip can detect the existence of 12 kinds of common causative substances: a kind of protozoon (Trichomonas vaginalis), 7 kinds of bacterium (chlamydia trachomatises, gonococcus, mycoplasma, mycoplasma hominis, Ureaplasma urealyticum, separates urea sporozoite, treponema pallidum) and 4 kinds of viruses (herpes simplex virus 1 & 2 type, human papillomavirus 6 type and 11 types).Whether amplify control (AC) has the DNA of the existence of inhibitor and extraction amount enough for monitoring in pcr amplification process.There is not expression in pcr amplification, have inhibitor or DNA quantity not sufficient (or there is not a corpse or other object for laboratory examination and chemical testing) in amplification control signal.Treponema pallidum (TP), can be provided as positive control (PC), and the positive template nucleic acid molecule that it contains is to monitor the performance of PCR reagent.Figure 10 b shows an example of venereal disease test kit array.
Figure 11 shows the embodiment of a thrombophilia test kit array.
Figure 12 shows the embodiment of a thrombophilia test kit array, and the annotation of its test result.
Summary of the invention
Following term is for describing the present invention.As do not set forth specific definitions, for describing its ordinary meaning that term of the present invention should give to be understood by those of ordinary skill.
As described herein, " equipotential specific oligonucleotide probe-reverse dot blot hybridization (ASO-RDB) " refers to the detection using and can be caught ASO-RDB target molecule in solids by hybridizing method.
" flow hybridization " refers to and utilizes at U.S. Patent number 5 as the term is employed herein, and 741, the crossover process of the technology described in 647.
" flow hybridization equipment " or " guiding device " refer at U.S. Patent number 6 as the term is employed herein, 020, equipment described in 187 and/or at PCT application WO/2011/139, the side direction fluidic device described in 750 or any guiding device designed subsequently.
detect pathogenic agent and target dna
Method provided by the invention, primer, probe and test kit are for detecting various nucleic acid, sudden change, material and pathogenic agent/or disease.In one embodiment, the present invention is applied to the existence (separately one or more) detecting tuberculosis, hepatitis B, hepatitis C, SARS, respiratory tract infection virus, sexual reverse, beta-globin, thrombophilia and/or related nucleic acid.The PCR of described method uses Auele Specific Primer to produce amplified production, described amplified production and oligonucleotide probe hybridization.Gained hybridization spectrum is by the existence of reflection nucleic acid, transgenation, material, pathogenic agent and/or disease.
In one embodiment, the hybridization of amplified production and oligonucleotide probe adopts method of river diversion, horizontal method of river diversion or reverse guide method.In one embodiment, the oligonucleotide probe for hybridizing is fixed on film.In the embodiment of method of river diversion, the susceptibility of hybridization depends on the total area of the film comprising described probe array.In the embodiment of horizontal method of river diversion, the susceptibility of hybridization depends on the total cross-sectional area of the film comprised on described probe leap flow direction.
In one embodiment, the invention provides a kind of method of the existence for detecting nucleic acid and/or allogenic material, comprise the following steps: (a) amplification is from the nucleic acid-templated nucleic acid molecule in Samples subjects, wherein primer is selected from sequence number and is: 174-181,201-205,241-244,263-286 and 299-306, to produce one or more amplified productions separately; (b) described amplified production and oligonucleotide probe hybridization, probe comprises and is selected from sequence number 182-200,206-240,245-260,287-298, and 307-314, produce hybridization spectrum with one or more oligonucleotide probes independent, illustrate whether target nucleic acid and/or allogenic material are present in Samples subjects.In one embodiment, the quantity of selected primer is 2-49; The number of selected probe be 2-92.In another embodiment, the quantity of selected primer be 2-24; The number of selected probe be 2-35.
In another embodiment, the invention provides a kind of method for detecting in sample whether there is nucleic acid and/or allogenic material, said method comprising the steps of: (a) amplification is from the template nucleic acid molecule in Samples subjects, wherein the primer is selected from sequence number and is: 174-181,201-205,241-244,263-286 and 299-306, to produce enough one or more amplified productions independent; (b) described amplified production and oligonucleotide probe hybridization, probe comprises and is selected from sequence number 182-200,206-240,245-260,287-298, and 307-314, produce hybridization spectrum with one or more oligonucleotide probes independent, illustrate whether target nucleic acid and/or allogenic material are present in Samples subjects.
In one embodiment, the invention provides a kind of for detecting the method for multi-drug resistant mycobacterium tuberculosis (DR-MTB) or its nucleic acid, described method comprises following steps: (a) obtains the sample comprising template nucleic acid molecule; B () uses and be selected from primer sequence number is template nucleic acid molecule described in the primer amplification of 174-181, thus obtains amplified production; (c) by the amplified production of gained be selected from the oligonucleotide probe hybridization that sequence number is 182-200, gained results of hybridization display multi-drug resistant tubercule bacillus (DR-MTB) or its nucleic acid whether exist.In one embodiment, the invention provides a kind of for detecting the method for multi-drug resistant mycobacterium tuberculosis (DR-MTB) or its nucleic acid, described method comprises following steps: (a) obtains the sample comprising template nucleic acid molecule; B () uses sequence number to be template nucleic acid molecule described in the primer amplification of 174-181, thus obtain amplified production; (c) be the oligonucleotide probe hybridization of 182-200 by the amplified production of gained and sequence number, whether gained results of hybridization display multi-drug resistant tubercule bacillus (DR-MTB) or its nucleic acid exist.In one embodiment, primer comprises the label that produces signal.In one embodiment, described amplified production by method of river diversion, horizontal method of river diversion or reverse guide method and/or relevant devices and multiple oligonucleotide probe hybridization.
In one embodiment, aforesaid method can detect the existence (DR-MTB) of multi-drug resistant mycobacterium tuberculosis, or has the nucleic acid of one or more DR-MTB following sudden change: one of seven sudden changes of rpoB gene; One of katG gene two sudden change; With a sudden change of inhA gene.Present invention also offers a kind of test kit, for detecting existence (DR-MTB) or its nucleic acid of multi-drug resistant mycobacterium tuberculosis, test kit comprise be selected from by sequence number be 182-200 primer and be selected from the oligonucleotide probe that sequence number is 174-181.In one embodiment, test kit is for detecting the existence (DR-MTB) of multi-drug resistant mycobacterium, or the nucleic acid of DR-MTB, and test kit comprises primer that sequence number is 174-181 and sequence number is the oligonucleotide probe of 182-200.
Present invention also offers the method detecting its betaglobulin of the experimenter sudden change suffering from β-thalassemia, comprise following steps: (a) obtains the sample comprising template nucleic acid molecule from experimenter; B () uses sequence number to be template nucleic acid molecule described in the primer amplification of 201-205, thus obtain amplified production; (c) by the amplified production of gained be selected from the oligonucleotide probe hybridization that sequence number is 206-240, gained results of hybridization display β-thalassemia genotype.In one embodiment, detect the method suffering from its betaglobulin transgenation of experimenter of β-thalassemia and comprise following steps: (a) obtains the sample comprising template nucleic acid molecule from experimenter; B () uses sequence number to be template nucleic acid molecule described in the primer amplification of 201-205, thus obtain amplified production; (c) be the oligonucleotide probe hybridization of 206-240 by the amplified production of gained and sequence number, the genotype of gained results of hybridization display β-thalassemia.In one embodiment, primer comprises the label that produces signal.Implement in disease at one, described amplified production by method of river diversion, horizontal method of river diversion, or reverse guide method and/or relevant devices and multiple oligonucleotide probe hybridization.In one embodiment, the method for above-mentioned detection betaglobulin sudden change can detect one of sudden change of 21 betaglobulins.
The present invention is also provided for the test kit detecting betaglobulin transgenation, and test kit comprises and is selected from primer that sequence number is 201-205 and is selected from the oligonucleotide probe that sequence number is 206-240.In one embodiment, be the primer of 201-205 and sequence number for the test kit that detects the sudden change of betaglobulin containing sequence number be the oligonucleotide probe of 206-240.
Present invention also offers a kind of method for detecting hepatitis B virus or its nucleic acid, step comprises: (a) obtains the sample comprising template nucleic acid molecule; B () uses and is selected from sequence number for template nucleic acid molecule described in the primer amplification of 241-244, thus obtain the amplified production of hepatitis B virus; (c) by the amplified production of gained be selected from the oligonucleotide probe hybridization that sequence number is 245-260, gained results of hybridization display hepatitis B virus or its nucleic acid whether exist.In one embodiment, the method for detecting hepatitis B virus or its nucleic acid comprises following steps: (a) obtains the sample comprising template nucleic acid molecule; B () uses sequence number to be template nucleic acid molecule described in the primer amplification of 241-244, thus obtain amplified production; (c) be the oligonucleotide probe hybridization of 245-260 by the amplified production of gained and sequence number, whether gained results of hybridization display hepatitis B virus or its nucleic acid exist.In one embodiment, primer comprises the label that produces signal.In one embodiment, described amplified production by method of river diversion, horizontal method of river diversion or reverse guide method and/or relevant devices and multiple oligonucleotide probe hybridization.
In one embodiment, aforesaid method can detect hepatitis B virus or its nucleic acid, and described hepatitis B virus is selected from genotype A to H.Present invention also offers a kind of test kit for detecting hepatitis B virus or its nucleic acid, comprising and being selected from primer that sequence number is 241-244 and sequence number is the oligonucleotide probe of 245-260.In one embodiment, the test kit for detecting hepatitis B virus or its nucleic acid comprises primer that sequence number is 241-244 and sequence number is the oligonucleotide probe of 245-260.
Present invention also offers a kind of method, for detecting experimenter's whether existence encephalapthy agent or its nucleic acid, described method comprises following steps: (a) obtains the sample comprising template nucleic acid molecule from described experimenter; B () uses and is selected from sequence number for template nucleic acid molecule described in the primer amplification of 263-286, thus obtain amplified production; (c) by the amplified production of gained be selected from the oligonucleotide probe hybridization that sequence number is 287-298, whether gained results of hybridization display sample exists Come from the nucleic acid of pathogenic agent that maybe to cause venereal disease.In one embodiment, the invention provides a kind of method, for detecting experimenter's whether existence encephalapthy agent or its nucleic acid, comprising following steps: (a) obtains the sample comprising template nucleic acid molecule from described experimenter; B () uses sequence number to be template nucleic acid molecule described in the primer amplification of 263-286, thus obtain amplified production; (c) be the oligonucleotide probe hybridization of 287-298 by the amplified production of gained and sequence number, whether gained results of hybridization display sample exists Come from the nucleic acid of pathogenic agent that maybe can cause venereal disease.In one embodiment, primer comprises the label that produces signal.In one embodiment, described amplified production by method of river diversion, horizontal method of river diversion or reverse guide method and/or relevant devices and multiple oligonucleotide probe hybridization.
In one embodiment, method for detecting sexual reverse in experimenter can detect following sexual reverse: Trichomonas vaginalis, chlamydia trachomatis, Diplococcus gonorrhoeae, mycoplasma genitalium, mycoplasma hominis, Ureaplasma urealyticum, separate sporozoite, treponema pallidum, herpes simplex virus type 1, herpes simplex virus type 2, human papillomavirus 6 type, and Human Papillomavirus Type 11. present invention also offers a kind of test kit of the existence for detecting sexual reverse or its nucleic acid in experimenter, described test kit comprise be selected from sequence number be 263-286 primer and be selected from the oligonucleotide probe that sequence number is 287-298.In one embodiment, the test kit for detecting sexual reverse or its nucleic acid in Samples subjects comprises primer that sequence number is 263-286 and sequence number is the oligonucleotide probe of 287-298.
The invention provides the method for detecting the transgenation relevant with thrombophilia, described method comprises following steps: (a) obtains the sample comprising template nucleic acid molecule; B () uses and is selected from sequence number for template nucleic acid molecule described in the primer amplification of 299-306, thus obtain amplified production; (c) by the amplified production of gained be selected from the oligonucleotide probe hybridization that sequence number is 307-314, gained results of hybridization display the transgenation relevant with thrombophilia whether exists.In one embodiment, described primer comprises the label that produces signal.In another embodiment, described method is for detecting the transgenation relevant with thrombophilia, and described method comprises following steps: (a) obtains the sample comprising template nucleic acid molecule; B () uses sequence number to be template nucleic acid molecule described in the primer amplification of 299-306, thus obtain amplified production; (c) be the oligonucleotide probe hybridization of 307-314 by the amplified production of gained and sequence number, whether the gained results of hybridization display transgenation relevant with thrombophilia exists.In one embodiment, described primer comprises the label that produces signal.In one embodiment, described amplified production by method of river diversion, horizontal method of river diversion, reverse guide method and/or relevant devices and multiple oligonucleotide probe hybridization.
In one embodiment, described method is for detecting the transgenation relevant with thrombophilia, described gene is selected from Leiden accelerator factor FactorVLeiden (FVL), thrombogen FactorII (FII or factor II), and Methylene tetrahydrofolate reductase MethylenetetrahydrofolateReductase (MTHFR).Present invention also offers the test kit for detecting the transgenation relevant with thrombophilia, described test kit comprise be selected from sequence number be 299-306 primer and be selected from the oligonucleotide probe that sequence number is 307-314.In one embodiment, described test kit comprises primer that sequence number is 299-306 and sequence number is the oligonucleotide probe of 307-314.
Experimental detail following by reference, the present invention may be better understood.But, it will be understood by those skilled in the art that provided embodiment is only as illustration, but not Xian System scope of the present invention.Scope of the present invention defined by claim subsequently.
In this application, refer to different reference or publication.The full text of these references or publication is all attached in the application with open, thus more fully describes situation of the present invention.It should be pointed out that Transitional Language " comprise " with ' comprise ' ﹑ ' containing ' or ' with ... for characteristic ' be synonym, comprising property or open, does not centrally get rid of the element or method steps that have and do not enumerate in addition.
Embodiment 1
test procedure
For DNA, pcr amplification, hybridization, detects the separating step of sample and interpretation of result, as WO/2011/139750 or be known as those skilled in the art and can be applied to the present invention in relevant program and technology.Various internal contrast (IC) can be included in described testing experiment, with validating DNA sample, and pcr amplification and result colour developing.The size of each water conservancy diversion membrane array and form, can be used for the needs according to individuality, or for optimizing the use of reaction chamber, to reach maximized cost-saving.
Embodiment 2
gene type multi-drug resistance tuberculosis
In one embodiment, the invention provides method and the test kit of DR-MTB gene type.Described method and test kit adopt polymerase chain reaction (PCR) and " water conservancy diversion " hybridization technique, for detecting the Mycobacterium tuberculosis (MTB) Rifampin (RIF) and vazadrine (INH) being had to resistance.For the rpoB gene that increases, the corresponding primer of katG gene and INHA gene has been proved does not have cross reaction with human genome.RS-IAC is an internal control primer, not for the mankind or MTB genome, for monitoring pcr amplification process.The transgenation of probe in detecting resistance used comprises rpoB gene (D516V, D516G, H526D, H526Y, H526L1, S531L and S531W), katG gene (S315T1 and S315T2), and inhA gene (-15TC/T).Also have five probes for detecting the sudden change of wild-type MTB, this contributes to checking rpoB gene, and whether the pcr amplification reaction of katG gene and inhA gene completes.In one embodiment, primer by biotinylation to hybridize.
In one embodiment, the invention provides the method for detecting multiple-drug resistance tuberculosis mycobacterium (DR-MTB), comprising the following steps: (a) obtains the sample comprising template nucleic acid molecule; (b) be selected from sequence number and be: the primer of 174-181, increase described template nucleic acid molecule, thus produce amplified production; (c) amplified production be selected from sequence number for: the group that the oligonucleotide probe of 182-200 forms hybridize, and gained hybridization spectrum will show the existence of multiple-drug resistance tuberculosis mycobacterium.In one embodiment, the present invention can detect multi-drug resistant mycobacterium tuberculosis, and has the existence of wherein one or more sudden changes following: seven sudden changes in rpoB gene; Two sudden changes in katG gene; A sudden change in inhA gene.In one embodiment, amplified production is by method of river diversion, horizontal method of river diversion, or reverse guide method, with the hybridization of multiple oligonucleotide probe.
Each position that Fig. 3 shows a DR-MTB case (left figure) and probe and signal (right figure) is embodied.IAC is that inner amplification controls, to monitor pcr amplification process.HC is that hybridization controls, monitoring crossover process.Fig. 4 shows one for detecting the embodiment of DR-MTB array configurations.In one embodiment, the MTB strain of resistance is had to be detected to RIF and vazadrine respectively.In another embodiment, the MTB bacterial strain of resistance (multi-drug resistant) is had to be detected to RIF and INH.In one embodiment, neither resistance to Rifampin (RIF), neither the MTB bacterial strain of resistance to vazadrine (INH) be detected.Detection comprises various control.
Primer
| Primer I D | Primer sequence (5'-3') | 5' modifies | Sequence number |
| MTB-DR-Rpob-F | TCAACATCCGGCCGGTGGTC | Vitamin H | 174 |
| MTB-DR-Rpob-R | CCGGCACGCTCACGTGACAGA | Vitamin H | 175 |
| MTB-DR-KatG-F | TGGCACCGGAACCGGTAAGGA | Vitamin H | 176 |
| MTB-DR-KatG-R | CGCCAGCAGGGCTCTTCGT | 177 | |
| MTB-DR-inhA-F | AAGTTCCCGCCGGAAATCGCAG | Vitamin H | 178 |
| MTB-DR-inhA-R | CCGGTAACCAGGACTGAACGGGAT | 179 | |
| RS-IAC-F | CGAGTTCCCGCCCATAACC | 180 | |
| RS-IAC-R | GGAAGTTGCAACGCCGTCC | Vitamin H | 181 |
Probe
Here is some for the example of PCR and hybridization:
| Composition | Capacity (μ L) |
| PCR pre-mixing | 19.6 |
| Primer pre-mixing | 2 |
| IAC | 0.1 |
| Taq polysaccharase | 0.4 |
| Nuclease free water | 0.9 |
| Template nucleic acid molecule | 2 |
| Amount to | 25.00 |
An embodiment of hybridization agreement:
Conditioning equipment temperature (downward)
Conditioning equipment temperature (rise)
Embodiment 3
β Mediterranean Sea gene type
The invention provides a kind of detection of betaglobulin transgenation of system, described transgenation comprises TATA-28 (A>G), TATA-29 (A>G), initiator codon (G>A), the detection of codon 5 (-CT), codon 8/9 (+G), codon 15 (G>A), codon 16 (-C), codon 17 (A>T), codon 19 (A>G), codon 26 (G>A) (Hb E), codon 27/28 (+three), codon is 30G>C, IVS1.1 (G>T), IVS1.1 (G>A), IVS1.5 (G>C), codon 41/42 (-TCTT), codon 43 (G>T), codon 71/72 (+A), IVS2.1 (G>A), the disappearance of IVS2.654 (C>T) and 619bp.
Present invention also offers the method detecting betaglobulin sudden change in Samples subjects, said method comprising the steps of: (a) obtains the sample comprising template nucleic acid molecule from experimenter; (b) be selected from group that primer that sequence number is: 201-205 forms and increase described template nucleic acid molecule, thus produce amplified production; (c) amplified production be selected from sequence number for: the group that the oligonucleotide probe of 206-240 forms hybridize, gained hybridization compose will show β-thalassemic genotype.In one embodiment, primer comprises a signal generation label.In one embodiment, amplified production is by method of river diversion, and horizontal method of river diversion, or reverse guide method, with multiple oligonucleotide probe hybridization.In one embodiment, aforesaid method ball is to detect any wherein one among 21 kinds of beta-globin sudden changes.
Fig. 5 shows the embodiment of a kind of β-thalassemia case and its signal location (in figure ruling only way for instructions).Fig. 6 a shows the genotypic example of different beta-thalassemia.Fig. 6 b illustrates the test result about using β-various clinical sample of thalassemia case, shows that the present invention can identify β-thalassemic genotype from human sample.
Primer sequence (number of adding AF007546.1):
Probe sequence (number of adding AF007546.1):
| Probe I D | Sense/antisense chain | 5' modifies | Probe sequence (5' to 3') | Sequence number |
| HBB2-A1 | Sense strand | Amido | AGACACCATGGTGCATCTG | 206 |
| HBB2-A1a | Sense strand | Amido | AGACACCATGGTGCACCT | 207 |
| HBB2-A2 | Sense strand | Amido | CAAACAGACACCAGGGTG | 208 |
| HBB2-A3 | Sense strand | Amido | GCTGGGCATAAAAGTCAGG | 209 |
| HBB2-A4 | Antisense strand | Amido | CCTGACTTCTATGCCCAGC | 210 |
| HBB2-A5 | Antisense strand | Amido | CCCTGACTTTCATGCCC | 211 |
| HBB2-B1 | Sense strand | Amido | GCATCTGACTCCTGAGGA | 212 |
| HBB2-B1a | Sense strand | Amido | GCACCTGACT CCTGAGG | 213 |
| HBB2-B2 | Antisense strand | Amido | ACTTCTCCTCGAGTCAGAT | 214 |
| HBB2-B3 | Antisense strand | Amido | CAGGGCCTCACCACCA | 215 |
| HBB2-B4 | Sense strand | Amido | GTTGGTGGTAAGGCCCT | 216 |
| HBB2-B5 | Antisense strand | Amido | CCAGGGGCCTCACCA | 217 |
| HBB2-C1 | Sense strand | Amido | AGGAGAAGTCTGCCGTTACT | 218 |
| HBB2-C2 | Sense strand | Amido | AGGAGAAGGTCTGCCGTTA | 219 |
| HBB2-C3 | Sense strand | Amido | GAGGTTCTTTGAGTCCTTTGG | 220 |
| HBB2-C4 | Antisense strand | Amido | CAAAGGACTCAACCTCTGG | 221 |
| HBB2-C5 | Sense strand | Amido | CCCAGAGGTTCTTTTAGTC | 222 |
| HBB2-D1 | Sense strand | Amido | TGTGGGGCAAGGTGAAC | 223 |
| HBB2-D2 | Sense strand | Amido | CCGTTACTGCCCTGTAGG | 224 |
| HBB2-D3 | Sense strand | Amido | CTGTGGGGAAGGTGAAC | 225 |
| HBB2-D4 | Antisense strand | Amido | TGTGGGGCTAGGTGAACG | 226 |
| HBB2-D5 | Antisense strand | Amido | CTTCATCCACGCTCACCT | 227 |
| HBB2-E1 | Antisense strand | Amido | TGATACCAACCTGCCCAG | 228 |
| HBB2-E2 | Sense strand | Amido | CTGGGCAGATTGGTATCA | 229 |
| HBB2-E3 | Sense strand | Amido | CCCTGGGCAGTTTGGTATC | 230 |
| HBB2-E4 | Sense strand | Amido | GCAGGTTGCTATCAAGGTTAC | 231 |
| HBB2-E5 | Antisense strand | Amido | ATACCAACGTGCCCAGG | 232 |
| HBB2-F1 | Sense strand | Amido | GGTGCCTTTAGTGATGGC | 233 |
| HBB2-F2 | Sense strand | Amido | TCGGTGCCTTTAAGTGATG | 234 |
| HBB2-F3 | Sense strand | Amido | TGGGTTAAGGCAATAGCAATAT | 235 |
| HBB2-F4 | Antisense strand | Amido | ATATTGCTATTACCTTAACCC | 236 |
| HBB2-G1 | Sense strand | Amido | CATACCTCTTATCTTCCTCC | 237 |
| HBB2-G2 | Sense strand | Amido | TGTAACAAGTAGAGATTCAAGTA | 238 |
| HBB2-G3 | Sense strand | Amido | GAACTTCAGGGTGAGTCTAT | 239 |
| HBB2-G4 | Antisense strand | Amido | GAACTTCAGGATGAGTCTATG | 240 |
Here is some for the exemplary protocols of PCR and hybridization:
| Composition | Capacity (μ L) |
| PCR pre-mixing | 19.6 |
| Taq polysaccharase | 0.4 |
| Template nucleic acid molecule | 2to 5.0* |
| Nuclease free water | Variable |
| Amount to | 25.00 |
An embodiment of hybridization agreement:
Data are read as the sudden change of β ball:
Embodiment 4
hepatitis B virus gene typing
Present invention also offers a kind of method for detecting hepatitis B virus, described method comprises step: (a) obtains the sample comprising template nucleic acid molecule; B () to be increased described template nucleic acid molecule with being selected from group that primer that sequence number is: 241-244 forms, thus produce amplified production; (c) amplified production be selected from sequence number for: the group that the oligonucleotide probe of 245-260 forms hybridize, and gained hybridization spectrum will show the existence of hepatitis B virus.In one embodiment, primer comprises a signal generation label.In one embodiment, amplified production is by method of river diversion, and horizontal method of river diversion, or reverse guide method, with multiple oligonucleotide probe hybridization.In one embodiment, general probe sequence number 257 is used to the nucleic acid of catching various genotypic hepatitis B virus.
The embodiment of 8 genotype of hepatitis B virus (A of hepatitis B virus, B, C, D, E, F, G and H type) that the Hepatitis B Virus Reagents box that Fig. 7 shows detects.Fig. 8 shows the example detecting different genotype of hepatitis B virus.
Primer sequence:
Probe sequence:
Hepatitis B virus positive control sequence [560bp] (pUC57 carrier):
TGGGATTCTATATAAGAGGGAAACTACACGTAGCGCCTCATTTTGCGGGTCACCATATTCTTGGGAACAAGAGCTACATCATGGGAGGTTGGTCATCAAAACCTCGCAAAGGCATGGGGACGAACCTTTCTGTTCCCAACCCTCTGGGATTCTTTCCCGATCATCAGTTGGACCCTGCATTCGGAGCCAATTCAAACAATCCAGATTGGGACTTCAACCCCATCAAGGACCACTGGCCACAAGCCAACCAGGTAGGAGTGGGAGCATTCGGGCCAGGGTTCACTCCCCCACACGGAGGTGTTTTGGGGTGGAGCCCTCAGGCTCAGGGCATATTGGCCACAGTGCCAGCAGTGCCTCCTCCTGCCTCCACCAATCGGCAGTCAGGAAGGCAGCCTACTCCCATCTCTCCACCTCTAAGAGACAGTCATCCTCAGGCCATGCAGTGGAATTCCACAGCTTTCCACCAAGCTCTGCAAGATCCCAGAGTCAGGGGCCTGTATTTTCCTGCTGGTGGCTCCAGTTCAGGAACACTCAACCCTGTTCCAACTATTGCCTCTCAC Serial number (261)
Internal control sequence [500bp] (pUC57 carrier):
GAGACAGGTTCGTCCAATCCCGTGCCGCGGCCTTGGCAGGGGGTTCGCAGGCCCCACCCGAAGCGTTGCTGAAGGCTCAGGCCTCTGAGCGACAAAAGCTTTAAACGCGAGTTCCCGCCCATAACCTGGACCGAATGCGGGACCATGCATCGTTCCACTGTGTTTGTCCCATGTAGGACGGGCGCAAGGCGTGCTTAGCTCAGCCTCGAATGCCTCGTATCATTGTGCACCCGCCGGTCACCAGCCAACGATGTGCGGACGGCGTTGCAACTTCCGGGGCCCAACCTGACCGTCCTGGGTACCGCACTCTGGGCAGTGCGAGGTAATGCCAGTCGCCCAGTGCCGAACAACACCTGACCTAACGGTAAGAGGCTCACATAATGGCTCCGCCGGCGCGCCCAGGGTACATTAGGTCAGCATCGGATGGACTGACATGAACCTTCACACCGAAGCGGAAACGGGTGCGTGGACCAGCGAGGAGCAAACGAAAATTCCTGGCC Serial number (262)
Below the example of some PCR agreements and hybridization agreement:
| Composition | Capacity (μ L) |
| The pre-mixing of hepatitis B virus primer | 19.6 |
| IAC | 0.1 |
| Taq polysaccharase | 0.3 |
| Template nucleic acid molecule | 5.0* |
| Nuclease free water | Variable |
| Amount to | 25.00 |
* advise total hepatitis B virus viral DNA in serum more than 4000IU/mL.
This temperature curve is applicable to Applied Biosystems, Inc.'s perkin elmer (ABI-PE) 9600, gene amplification PCR system 9700, Veriti (AppliedBiosystems company), PTC-200 (MJ research)..
An embodiment of hybridization agreement:
Embodiment 5
gene type, and detect multiple sexual reverse simultaneously
In one embodiment, the invention provides and detect the method for sexual reverse, described pathogenic agent including, but not limited to, protozoon, bacterium and virus.In one embodiment, sexual reverse is present in urine, urogenital system swab (urethra, vagina, uterine cervix and damage) and liquid based cytology sample (PreservCyt
tMand SurePath
tM).In one embodiment, amplification control (AC) comprises the material for detecting human DNA.With pcr amplification urine urogenital system swab (urethra, vagina, uterine cervix and damage) and liquid based cytology sample (PreservCyt
tMand SurePath
tM) in the target DNA that extracts, then by hybridization, reach to quicker, sensitiveer and detect venereal disease more specifically.In one embodiment, the sexual reverse that described method 12 kinds of can detect are common, comprise a kind of protozoon (Trichomonas vaginalis), 7 kinds of bacterium (chlamydia trachomatis, Diplococcus gonorrhoeae, mycoplasma genitalium, mycoplasma hominis, Ureaplasma urealyticum, separates sporozoite, treponema pallidum) and 4 kinds of viruses (herpes simplex virus type 1 & 2 type, human papillomavirus 6 type and 11 types).These pathogenic agent all with trachelitis, urethritis, trichomoniasis is relevant with pelvic inflammatory disease.In another embodiment, include universal primer (S) in test and/or amplify and control.
Present invention also offers a kind of method, for detecting the sexual reverse existed in Samples subjects, the step of described method comprises: (a) obtains the sample comprising template nucleic acid molecule from experimenter; B group that () forms by being selected from primer that sequence number is: 263-286, increase described template nucleic acid molecule, thus produce amplified production; (c) amplified production be selected from sequence number and be: the oligonucleotide probe hybridization of 287-298, gained hybridization spectrum will show nucleic acid fragment and derive from sexual reverse.In one embodiment, primer comprises a signal generation label.In one embodiment, be a method of river diversion with the hybridization of the amplified production of multiple oligonucleotide probe, horizontal method of river diversion or reverse guide method and/or relevant devices.
The primer of sequence number 263-284 is made up of two portions: 5' part is the nucleotide sequence of synthetic, and target pathogen not homology, does not also relate to the sequence of any organism can be able to found in the mankind; 3' part is pathogen specific sequence, makes primer effectively can be attached to the nucleic acid of corresponding pathogenic agent, to produce amplified production.The 5' part of all primer sequences is universal sequence.Universal primer sequence design be through careful consideration and experiment, the other biological body in amplified sample can be got rid of, any thymus nucleic acid (Fig. 1) of such as other pathogenic agent and human genome.Such as those are disclosedly in the present invention verified as specific universal sequence by selection and then merge with various pathogen specific sequence, and obtain pathogen specific primer, it can produce the amplified production comprising universal sequence.These amplified productions become the template nucleic acid molecule of second time PCR, and then use sequence number to be: the universal primer of 285 and 286 to increase all template nucleic acid molecules with same efficiency.Therefore the amplified production ultimate density meeting of linear generation and the concentration of initial target DNA are directly proportional.Even if target copy number is low in sample, still can amplifies to reach suitable concentration and detect the positive again, use universal primer to correct a mistake, significantly improve sensitivity.In one embodiment, the PCR reaction of two steps is carried out in single test tube; Pathogen specific primer is wherein attached to target molecule by its gene specific 3' region, to produce the primary amplification product containing 5' universal sequence.These amplified productions as the template nucleic acid molecule of described second group of primer (universal primer), are detected by hybridization with the amplified production produced containing signal.Fig. 2 illustrates the embodiment of an amplification scheme.
Because the present invention uses universal primer, therefore than other sexual reverse test kits on market detection method more accurately, sensitiveer.Just as illustrated in fig. 9, the present invention can detect and be low to moderate 50-300 sexual reverse copy number, and the detection of another makers' test kit at least will have 500 sexual reverse copy numbers; Therefore the present invention can detect the sample of lower pathogenic agent concentration.
Fig. 9 b shows the Performance comparision of other venereal disease check reagent boxes on the present invention and market.Result confirms four tests and actual gene type by quantitative PCR (qPCR).After confirming the genotype of pathogenic agent, the present invention's (as tested 3 and 4 in figure) is more less than other test kits there is error result (as tested 1 and 2 in figure).In addition, the present invention can detect multiple pathogens (going out as shown in figure 9b), and therefore, compared with other test kits on market, it is the highest number that the present invention detects 12 kinds of pathogenic agent simultaneously.The sickness rate infected after HPV6 and HPV11 is very high, and therefore the covering of the present invention to HPV6 type and 11 types is very useful.Genital warts is a kind of venereal disease with hyperinfection power, and wherein the case of 90% is owing to infecting caused by HPV6 and HPV11.
Figure 10 a shows the embodiment that is detected venereal disease array, array can detect the existence of 12 kinds of common venereal diseases pathogenic agent: a kind of protozoon (Trichomonas vaginalis), 7 kinds of bacterium (chlamydia trachomatises, Diplococcus gonorrhoeae, mycoplasma genitalium, mycoplasma hominis, Ureaplasma urealyticum, separate sporozoite, treponema pallidum) and 4 kinds of viruses (herpes simplex virus type 1 and 2 types, human papillomavirus 6 type and 11 types).Amplify control (AC) and illustrate that whether result is effective.Figure 10 b shows the example that is explained venereal disease array.In one embodiment, protozoon, bacterium and virus are measured separately.In another embodiment, protozoon, bacterium and virus (multiple infection) are detected simultaneously.
In one embodiment, the template nucleic acid molecule of treponema pallidum (TP) is provided as the performance of positive control (PC) for monitoring PCR reagent.If there is not positive control (PC) signal of TP in a test, this may be because described DNA because of long storage periods or nuclease contaminated and degenerate.But if the positive signal that test sample produces is any pathogenic agent, detected result remains effective.In the test of positive control (PC), because positive control sample only comprises treponema pallidum (TP), but there is no endogenic human gene group DNA, will cause lacking AC signal.Show that contrast test is invalid in the test of the existence (PC) of the AC signal of positive control, this may be should described repetition owing to polluting and testing.
As AC signal does not occur, this expression is tested the DNA quantity not sufficient in sample or does not exist.But if test sample produces the positive signal of any pathogenic agent, test remains effective.Owing to may there be the competition from other pathogenic agent DNA, in some samples, AC signal may not occur.In this case, as long as array there is one or more positive signal, detected result remains effective.
In another embodiment, the array box used in water conservancy diversion process and/or device have different forms and size.Use outside the array of 10 points, also can use 35 lattice arrays, also can to add array point again for the transgenation of test example as extra pathogen gene hypotype and/or Resistant strain.Detection signal is also by using the image system of adjoint FT-Pro hybridization device to reach to digitizing.In another embodiment, hybrid device used is an automated installation, has binding analysis and digitizing function.
The present invention than detection method existing on market and test kit more convenient, can increase because of the present invention simultaneously and detect multiple target, there is highly sensitive and contain 12 kinds of sexual reverse, comprising by the excluded HPV subgenotype of most of available reagent box on market.By connecting with flow hybridization, the present invention allows rapid sensitive and high-throughout sexual reverse to detect.
Primer sequence:
Probe sequence:
| Cause of disease/target | Probe | Probe sequence (5 ' to 3 ') | 5' modifies | Sequence number |
| CT | CT_IN1 | AGTGCATAAACTTCTGAGG | Amido | 287 |
| NG | NG_Pr2 | TATAAACGCCCGGCAGTTAC | Amido | 288 |
| MG | MG_IN2 | AAAGATTACTGGAGAGAACC | Amido | 289 |
| UU | UU_Pr | TGAACGAAGGTAGAGAAGCA | Amido | 290 |
| UP | UMPr | TGCGGTTTACGAAATTGAAA | Amido | 291 |
| TV | TVKSp | ACTCATGACGAACGAAGAAG | Amido | 292 |
| TP | TP_IN2 | GCAGAAAAACTATCCTCAG | Amido | 293 |
| MH | MHPr | CAGCTATGCTGCGGTGAATA | Amido | 294 |
| HSV1/2 | HSV_Pr1(minus strand) | CTTGTCGATCACCTCCTCG | Amido | 295 |
| HPV6 | HPV6_IN2 | TTATGTGCATCCGTAACTA | Amido | 296 |
| HPV11 | hPV11_Pr(minus strand) | GTAGCAGATTTAGACACAGATG | Amido | 297 |
| AC | b-globin-402T | AAGGTGAACGTGGATGAAGTTGGTGG | Amido | 298 |
Here is some example PCR and hybridization agreement:
| Composition | Capacity (μ L) |
| PCR pre-mixing | 18.6 |
| The pre-mixing of venereal disease primer | 1.0 |
| DNA Taq polysaccharase | 0.4 |
| Venereal disease DNA positive regulation | Maximum 5.0* |
| Nuclease free water | Variable |
| Amount to | 25.0 |
* the scope of the suggestion of STb gene: 5ng – 100ng.
Above thermocycling program is suitable for
thermalCycler (Lifetechnologies) and S1000
tMthermalCycler (Bio-rad). above thermocycling program can for other thermal cycler (ramp rate reaches 3 DEG C/sec) after amendment.
An embodiment of hybridization agreement:
Embodiment 6
the related gene somatotype of thrombophilia
Present invention also offers the method for detecting the transgenation relevant with thrombophilia, described method comprises following steps: (a) obtains the sample comprising template nucleic acid molecule; B () uses and is selected from sequence number for template nucleic acid molecule described in the primer amplification of 299-306, thus obtain amplified production; (c) by the amplified production of gained be selected from the oligonucleotide probe hybridization that sequence number is 307-314, gained results of hybridization display the transgenation relevant with thrombophilia whether exists.In one embodiment, described primer comprises the label that produces signal.In one embodiment, described amplified production is by method of river diversion, horizontal method of river diversion or reverse guide method and multiple oligonucleotide probe hybridization.In one embodiment, aforesaid method can detect and be selected from Leiden accelerator factor FactorVLeiden (FVL), thrombogen FactorII (FII or factor II), and the transgenation of Methylene tetrahydrofolate reductase MethylenetetrahydrofolateReductase (MTHFR).
Figure 11 shows the embodiment of a thrombophilia test kit array.Figure 12 shows an annotation example of its test result of thrombophilia test kit.
Primer sequence:
| Primer | Primer sequence (5'-3') | 5' modifies | Sequence number |
| FVL_F | GAAAATGATGCCCAGTGCTT | / | 299 |
| FVL_R | TTGAAGGAAATGCCCCATTA | Vitamin H | 300 |
| FII_F | GAACCAATCCCGTGAAAGAA | / | 301 |
| FII_R | AGCTGCCCATGAATAGCACT | Vitamin H | 302 |
| MTHFR677_F | GGTTACCCCAAAGGCCACC | Vitamin H | 303 |
| MTHFR677_R | AAGCGGAAGAATGTGTCAGC | Vitamin H | 304 |
| MTHFR1298_F | TTTGGGGAGCTGAAGGACTA | / | 305 |
| MTHFR1298_R | CTTTGTGACCATTCCGGTTT | Vitamin H | 306 |
Probe sequence:
Reaction mixture:
| Composition | Capacity (μ L) |
| Thrombophilia PCR pre-mixing | 21.6 |
| The pre-mixing of Thrombophilia primer | 1.0 |
| Enzyme mixation | 0.4 |
| Template nucleic acid molecule | Maximum 2.0 |
| Nuclease free water | Variable |
| Amount to | 25.00 |
* the scope of the suggestion of DNA total amount: 10ng – 100ng.
An embodiment of hybridization procedures:
Claims (20)
1., for detecting a method for experimenter's whether existence encephalapthy agent, comprise following steps:
A () obtains the sample comprising template nucleic acid molecule from described experimenter;
B () uses sequence number to be template nucleic acid molecule described in the primer amplification of 263-286, thus obtain amplified production; With
C the amplified production of gained and sequence number are the oligonucleotide probe hybridization of 287-298 by (), gained results of hybridization display experimenter whether existence encephalapthy agent.
2. method according to claim 1, wherein said amplified production is by method of river diversion, horizontal method of river diversion or reverse guide method and multiple oligonucleotide probe hybridization.
3. method according to claim 1, wherein said method can detect and be selected from Trichomonas vaginalis, chlamydia trachomatis, Diplococcus gonorrhoeae, mycoplasma genitalium, mycoplasma hominis, Ureaplasma urealyticum, solution sporozoite, the sexual reverse of treponema pallidum, herpes simplex virus type 1, herpes simplex virus type 2, human papillomavirus 6 type and Human Papillomavirus Type 11.
4., for detecting a test kit for sexual reverse, comprise primer that sequence number is 263-286 and sequence number is the oligonucleotide probe of 287-298.
5., for detecting a method for multi-drug resistant mycobacterium tuberculosis or its nucleic acid, comprise following steps:
A () obtains the sample comprising template nucleic acid molecule;
B () uses sequence number to be template nucleic acid molecule described in the primer amplification of 174-181, thus obtain amplified production; With
C the amplified production of gained and sequence number are the oligonucleotide probe hybridization of 182-200 by (), whether bacterium exists gained results of hybridization display multiple-drug resistance tuberculosis.
6. method according to claim 5, wherein said amplified production is by method of river diversion, horizontal method of river diversion or reverse guide method and multiple oligonucleotide probe hybridization.
7. method according to claim 5, wherein said method can detect the multi-drug resistant mycobacterium tuberculosis with one or more nucleic acid mutations following:
One of seven sudden changes of rpoB gene;
One of two sudden changes of katG gene; With
A sudden change of inhA gene.
8., for detecting a test kit for multi-drug resistant mycobacterium tuberculosis, comprise primer that sequence number is 174-181 and sequence number is the oligonucleotide probe of 182-200.
9., for detecting the method suffering from β-its betaglobulin of thalassemia experimenter sudden change, comprise following steps:
A () obtains the sample comprising template nucleic acid molecule from described experimenter;
B () uses sequence number to be template nucleic acid molecule described in the primer amplification of 201-205, thus obtain amplified production; With
C the amplified production of gained and sequence number are the oligonucleotide probe hybridization of 206-240 by (), the genotype of gained results of hybridization display experimenter β-thalassemia.
10. method according to claim 9, wherein said amplified production is by method of river diversion, horizontal method of river diversion or reverse guide method and multiple oligonucleotide probe hybridization.
11. methods according to claim 9, wherein said method can detect one of sudden change of 21 betaglobulins.
12. 1 kinds, for detecting the test kit of betaglobulin sudden change, comprise primer that sequence number is 201-205 and sequence number is the oligonucleotide probe of 206-240.
13. 1 kinds, for detecting the method for hepatitis B virus, comprise following steps:
A () obtains the sample comprising template nucleic acid molecule;
B () uses sequence number to be template nucleic acid molecule described in the primer amplification of 241-244, thus obtain amplified production; With
C the amplified production of gained and sequence number are the oligonucleotide probe hybridization of 245-260 by (), whether hepatitis B virus exists the display of gained results of hybridization.
14. methods according to claim 13, wherein said amplified production is by method of river diversion, horizontal method of river diversion or reverse guide method and multiple oligonucleotide probe hybridization.
15. methods according to claim 13, wherein said method can detect the hepatitis B virus being selected from genotype A to H.
16. 1 kinds, for detecting the test kit of hepatitis B virus, comprise primer that sequence number is 241-244 and sequence number is the oligonucleotide probe of 245-260.
17. 1 kinds, for detecting the method for the sudden change relevant with thrombophilia, comprise following steps:
A () obtains the sample containing template nucleic acid molecule nucleic acid molecule;
B () uses sequence number to be template nucleic acid molecule described in the primer amplification of 299-306, thus obtain amplified production; With
C the amplified production of gained and sequence number are the oligonucleotide probe hybridization of 307-314 by (), whether the gained results of hybridization display sudden change relevant with thrombophilia exists.
18. methods according to claim 17, wherein said amplified production is by method of river diversion, horizontal method of river diversion or reverse guide method and multiple oligonucleotide probe hybridization.
19. methods according to claim 17, wherein said method can detect and be selected from Leiden accelerator factor (FactorVLeiden), the transgenation of thrombogen (FactorII) and Methylene tetrahydrofolate reductase (MethylenetetrahydrofolateReductase).
20. 1 kinds, for detecting the test kit of the sudden change relevant with thrombophilia, comprise primer that sequence number is 299-306 and sequence number is the oligonucleotide probe of 307-314.
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| CN109457037A (en) * | 2018-10-24 | 2019-03-12 | 美林美邦（厦门）生物科技有限公司 | A kind of genital tract causal agent kit for detecting nucleic acid |
| CN113416743A (en) * | 2021-07-26 | 2021-09-21 | 广东省农业科学院动物卫生研究所 | Nucleic acid molecule, PCR primer pair and kit for detecting avian trichomonas rpoB gene |
| RU2778666C1 (en) * | 2021-09-27 | 2022-08-22 | Федеральное государственное бюджетное образовательное учреждение высшего образования "Смоленский государственный медицинский университет" министерства здравоохранения Российской Федерации | METHOD FOR DETECTING MUTATIONS IN THE parC AND gyrA QRDR GENE REGIONS LEADING TO RESISTANCE IN MYCOPLASMA GENITALIUM TO FLUOROQUINOLONE ANTIBIOTICS |
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| AP2017009731A0 (en) | 2014-07-24 | 2017-02-28 | Abbott Molecular Inc | Compositions and methods for the detection and analysis of mycobacterium tuberculosis |
| WO2016057905A1 (en) * | 2014-10-10 | 2016-04-14 | Rutgers, The State University Of New Jersey | Polymerase chain reaction primers and probes for mycobacterium tuberculosis |
| CN106290884A (en) * | 2015-06-15 | 2017-01-04 | 江苏戴格诺思生物技术有限公司 | A kind of Ureaplasma urealyticum gold mark detection kit and detection method |
| WO2017011565A1 (en) * | 2015-07-14 | 2017-01-19 | Abbott Molecular Inc. | Compositions and methods for identifying drug resistant tuberculosis |
| US11015228B2 (en) * | 2018-01-09 | 2021-05-25 | Abbott Molecular Inc. | Assay for detecting Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, and Mycoplasma genitalium |
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| CN108660254B (en) * | 2018-05-30 | 2023-07-28 | 杭州千基生物科技有限公司 | Primer, probe, kit and method for detecting genital tract pathogen nucleic acid |
| CN109487014B (en) * | 2019-01-09 | 2022-08-02 | 中生方政生物技术股份有限公司 | Herpes simplex virus II type detection marker, primer probe pair, kit and detection method |
| CN111593112A (en) * | 2020-05-12 | 2020-08-28 | 深圳市星蝶科技有限公司 | PCR reagent and kit for detecting beta-thalassemia |
| RU2750715C1 (en) * | 2020-12-23 | 2021-07-01 | Федеральное государственное бюджетное образовательное учреждение дополнительного профессионального образования "Российская медицинская академия непрерывного профессионального образования" Министерства здравоохранения Российской Федерации (ФГБОУ ДПО РМАНПО Минздрава России) | Method for determining the genetic predisposition to the development of multidrug-resistant tuberculosis mycobacterium tuberculosis in hiv infection |
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| CN113416743B (en) * | 2021-07-26 | 2023-06-16 | 广东省农业科学院动物卫生研究所 | Nucleic acid molecule, PCR primer pair and kit for detecting trichomonas fowl rpoB gene |
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Also Published As
| Publication number | Publication date |
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| CN106906306B (en) | 2020-07-10 |
| CN106906306A (en) | 2017-06-30 |
| WO2014139330A1 (en) | 2014-09-18 |
| HK1217515A1 (en) | 2017-01-13 |
| CN105392896B (en) | 2017-10-20 |
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