CN105392896B - Quick and sensitive genotype identification and detection of nucleic acids - Google Patents
Quick and sensitive genotype identification and detection of nucleic acids Download PDFInfo
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- CN105392896B CN105392896B CN201480027795.3A CN201480027795A CN105392896B CN 105392896 B CN105392896 B CN 105392896B CN 201480027795 A CN201480027795 A CN 201480027795A CN 105392896 B CN105392896 B CN 105392896B
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Abstract
The invention discloses method, primer, probe and the kit for identifying various gene mutations or substance gene parting.In one embodiment, the material is pathogenic.In another embodiment, the present invention is applied to detection multi-drug resistant mycobacterium tuberculosis, hepatitis B, beta Globulin mutation, the mutation relevant with thrombophilia；Or a variety of sexual reverses, including but not limited to, trichomonas vaginalis, Chlamydia, gonococcus, mycoplasma, mycoplasma hominis, Ureaplasma urealyticum, solution sporozoite, microspironema pallidum, herpes simplex virus type 1 and 2 types and the type of HPV 6 and 11 types.
Description
Related application
It is priority base this application claims the U.S. Provisional Patent Application No. 61/791,933 submitted with March 15th, 2013
Plinth, all the elements of the patent application are all included into the application by reference.
Technical field
Field of the present invention is to identify the various genotype relevant with human diseases.
Background technology
The present invention relates to identification different genotype.Sequence specific primers-PCR (SSP-PCR), DNA are surveyed
Sequence, DNA fingerprint, and the technology such as SNP (SNP) Genotyping, have been applied to Genotyping (PCT Application Publication
Number WO/2011/139750).In these techniques, SNP (SNP) Genotyping has higher discernment.So
And, most of Genotyping detections for being related to DNA hybridization require higher operating cost and longer operating time, therefore have
The more effective methods of genotyping of necessity exploitation, reach faster, less expensive and covering more polygene type.
Mycobacterium tuberculosis (MTB) is detected
Tuberculosis (TB) is by caused by mycobacterium tuberculosis (Mycobacterium tuberculosisMTB).Resistance
The appearance of property mycobacterium tuberculosis (DR-MTB) is one in industrialized country and developing country's Tuberculosis
Serious problems.Except the importance on clinical and epidemiology, drug-resistant tuberculosis (DR-MTB) more has important economic shadow
Ring, because of its treatment cost than common MTB to be high.DR-MTB is defined as having resistance to at least two maximally effective First Line medicines
The property of medicine, including rifampin (rifampinRIF) and isoniazid (isoniazidINH).DR-MTB cases have becoming for rising in the whole world
Gesture, significant morbidity and mortality.The drug resistance of one line antituberculotic has been observed that relevant with several MTB gene mutations：
RpoB (resistance to RIF)；KatG genes and inhA genes (resistance to INH).
DR-MTB detection has several different technologies can use.Although traditional phenotypic drug sensitivity experiment (DST) is still
It is test MTB drug resistance " Yellow goldstandards ", but DST may be up to could complete in eight weeks.Mycobacterium tuberculosis is raw
Long slow bacterium.MTB just divided once per 15-20 hours, and generally required 4 to 6 all ability depending on used medium
Grow into bacterium colony.Hereafter, when the DST MTB cultivated are identified, drug sensitive test generally also takes 2 weeks again.DST therefore need compared with
For a long time, the delay for the treatment of disease is result in, especially common practice is to start treatment before DST completions.Industry is opened
The time needed for test is cultivated or can shortened to replacement in hair；However, the MTB in some type of sample is not suitable for replacement training
Support.In business environment and conventional DNA sequencing is not frequently used to detect MTB medicament-resistant mutations, because it is time-consuming and need special
Knowledge.In the market has a kit of different technologies, including real-time PCR and for detecting single type MTB's or drug resistance MTB
Conventional hybridization.Particularly real-time PCR, because its number of channels available is limited, so being generally difficult to while detecting a variety of resistances
MTB.Although conventional hybridization is also applied for detection multidrug resistance MTB；However, it needs long incubative time (at least 4 hours).It is right
Than under, the present invention by using polymerase chain reaction (PCR) and " water conservancy diversion " hybridization technique there is provided a kind of array test, can be with
Detect single resistance MTB and multidrug resistant MTB simultaneously on a platform, detection time is shorter (~35 points than conventional hybridization
Clock).DNA need to be only less than 4 hours to the whole test of analysis from sample is extracted, and testing result has the specific and sensitivity of height
Property.
The detection of beta Thalassemia
Beta Thalassemia is caused by autosome mutation, is a kind of hemoglobin disease, can be according to the production of betaglobulin
Impacted degree is classified again.Some of which mutation causes beta Globulin (to be expressed as β+) the slight underproduction, some cause betaglobulin
The absolute silence of gene and can not produce or only produce and lose the beta Globulin of function and (be expressed as β0).It is estimated that the world there are about
1.5% population has a heterozygote of β-thalassemia genotype, and father and mother are that the Words of heterozygote its offspring is probably homozygote.
Beta Thalassemia has three orders of severity：Slightly (minor thalassemia, β+/ β or β0/ β), moderate (thalassemia intermedia：
β+/ β or β0/ β) and serious (major thalaseemia：β+/β+, β0/β+Or β0/β0).The people of heredity thalassemia mainly can
There is hypochrosis microcytic anemia, and lifelong blood transfusion may be needed.The best method for controlling β-thalassemia is to avoid β Mediterranean poor
The birth of blood infant, this wants quasi- father and mother to do antenatal detection and genetic counselling；This has been found to be most effective management thalassemia
Method.β-thalassemia is popular in Temperate Region in China, such as Mediterranean country, the Middle East and south east asia.β-Mediterranean
The molecular basis of anaemia has been widely studied, and finds the point mutation of betaglobulin gene and has different mutation in different areas
Mode and the frequency of mutation.
Current detection technique includes microarray, ApoE gene (AS-PCR) and direct Sequencing.Microarray can
To detect on multiple target dna chips simultaneously, therefore suitable for detecting multiple samples simultaneously.However, its application is Yin Qigao
The limitation of cost, as a result can not be detected by an unaided eye, and high-performance scanner and complicated analysis software need to be used to explain.Equipotential
Gene specific PCR is to use allele-specific primers, is only had and target in polymorphism or mutation , zhe technologies to detect
The oligonucleotides that DNA is matched completely potentially acts as primer to expand target dna.The limitation of the technology be one reaction it is limited
Handling capacity.Direct DNA sequencing is related to external DNA replication dna process wherein archaeal dna polymerase and optionally mixes chain termination double deoxidation
Nucleotides, the handling capacity of the technology is low and needs long-time operation, therefore detection efficiency is low.On the other hand, the invention provides
Yi Zhong Knot close the DNA detections using PCR and reverse dot blot hybridization, can Enough provide the β of an extremely sensitive and special detection-
The mutation of globulin gene, to assist the detection of β-thalassemia.
Hepatitis type B virus detects
Hepatitis type B virus is minimum DNA virus, and it includes 3000 nucleotides surrounded by Protein capsid.It is B-mode
Hepatitis viruse with the blood or bioresorbable of sufferer by propagating.About 2,000,000,000 people infect hepatitis B；Wherein more than 3.5
Hundred million turn into hepatitis carrier.About 1/5th hepatitis b virus infected can develop hepatic sclerosis or liver cancer.Detect second
Hepatitis virus can be by simple surface antigen blood testing, or uses application polymerase chain reaction (PCR) and B-mode liver
The measurement of scorching viral DNA is diagnosed.In addition, having identified 10 kinds by analyzing the difference of HBV genomic sequence
Genotype of hepatitis B virus (A to J) and several hypotypes.Genotype of hepatitis B virus and hypotype substantially show different ground
Domain is distributed.Various genotype of hepatitis B virus have differences on causing a disease and treating.The determination of genotype of hepatitis B virus
Information can be provided for the assessment before chronic HBV infection and treatment.DNA sequencing is considered as to hepatitis B
Virus gene type detects Yellow goldstandard methods.But it is less efficient detection mixing genotype.In addition, molecular dna technology is carried
A specific method for being used to detect genotype of hepatitis B virus extremely sensitively is supplied.
Sexual reverse is detected
Venereal disease is common worldwide disease.Estimate there are 448 Wan Xinfa every year and can according to the World Health Organization in 2005
The sexually transmitted diseases (syphilis, gonorrhoea, Chlamydia and trichomonad) of healing, occur mainly in the adult of 15 to 49 years old all over the world.
In 2010, every 550 pregnant woman had one to be detected to suffer from sexually transmitted disease in Hong Kong (not including AIDS).Venereal disease can be infected
The child of pregnant woman, either in period of gestation or after baby is born.Central, gonococcus and Chlamydia can cause infertility.Therefore,
Quasi- wedding Mr. and Mrs and pregnant preceding women have the potential demand of detection venereal disease.In addition, China is also the potential market for detecting sexual reverse,
Sharply increased in past 20 years std patient quantity.Therefore China needs quick, low price, accurate sexual reverse check reagent box
Help controls venereal disease.Venereal disease check reagent box in the market can be used for detecting that single pathogen Huo Tong Time detect 3-4 cause of disease
Body, it is typically no including HPV (HPV).It is in need while screening analysis due to the incidence of disease increase of venereal disease
With the multiple sexual reverses of detection.
Thrombophilia is detected
Thrombophilia refers to that blood has abnormal solidification, adds the thrombotic risk of appearance.Research report shows
Show have in the thrombotic patients of thrombophilia Bing Chu Now, about 40% With inherent causes are relevant, and have been proved higher
Risk suffers from the dysgenesias such as the angiocardiopathies such as venous thronbosis and recurrent miscarriage.Also increasing opinion thinks
The pregnant woman for the thrombophilia that is hereditary is easier to pregnancy badness occur, and symptom includes recurrent miscarriage, Intrauterine Fetal Death, fetus
Intrauterine growth retardation, aura eclampsia and placental abruption etc..
The past Er Shi Inter of, some gene mutations have been determined relevant with heredity thrombophilia.It is most common
Four gene mutations be：Leiden accelerator factor Factor V Leiden (FVL) [1691G>A], factor Factor
II (FII or for factor II) [20210G>A], methylenetetrahydrofolate reductase
Methylenetetrahydrofolate Reductase(MTHFR)[677C>T] and methylenetetrahydrofolate reductase
Methylenetetrahydrofolate Reductase(MTHFR)[1298A>C].Detect that these gene mutations contribute to knowledge
There is not the people at highest risk of thrombophilia and recurrent miscarriage, so as to help Ge Do personages prevention disease and the ill wind of reduction
Danger, and preventative-therapeutic guidance is provided.
Brief summary of the invention
SNP Genotypings are used as diagnostic tool
SNP Genotypings can be also used for identification genetic fragment, Genetic polymorphism, have in addition to available for DNA fingerprint
The gene of change, or the problematic gene of function.The method provided by the present invention and kit are used for quick, clearly identification tool infection
Property pathogen, due to specific dna sequence presence or missing caused by genetic disease.
Discussed as more than, the hybridization format of in the market is needed by having high solution as the image analyzer of degree is analyzed.With film
As basis equipotential specific oligonucleotide probe-reverse dot blot hybridization (ASO-RDB) flow hybridization form (such as U.S. Patent number 5,
741,647) SNP Genotypings can be applied to.Microarray hybridization form can produce visible point, it is possible to by range estimation and/
Or the relatively low image analyzer of use cost is analyzed.
Flow hybridization
DNA hybridization is always modern biotechnology scientific research and is related to the most important method in gene molecule biological study.
The complicated performance of the nucleotide sequence of various genomes is hybridized in the solution by the DNA sequence dna of particular complementary to be revealed and analyzes.Letter
Yan Zhi, target dna is amplified or digested, and it is visited with the complementary DNA on solid support matrix (such as nitrocellulose filter)
Pin hybridizes.However, traditional hybridization technique is limited to the area of film in principle, and usually require longer incubative time.
Flow hybridization method and apparatus can control hybridization conditions exactly, do not have conventional hybridization technology take it is longer
Wen Questions.Hybridization time from a few hours or a couple of days can be reduced to several minutes by water conservancy diversion DNA hybridization technology, and (whole hybridization assays can be in 5-
Completed in 30 minutes, the specific time depends on the method for being used to produce detection signal).The manufacturing cost of flow hybridization equipment is low
It is honest and clean, and using 10 times fewer than conventional hybridization equipment of amount of reagent, therefore make DNA detection techniques than preceding more economical material benefit.Water conservancy diversion
Hybridization technique provides sensitiveer, accurate detection and qualification result, and is generally applicable to various different technologies such as tradition DNA prints
Mark method (Southern blotting), RNA blottings (Northern blotting), dot blotting, slit blotting and
The hybridization of reverse dot blotting.
PCT application WO/2011/139750 describes the multiple horizontal fast guiding inspections for being connected to central control unit
Measurement equipment.Hybridization device includes a central control unit for being connected to one or more lateral flow devices.Lateral flow device carries out miscellaneous
Friendship process and developing programs, and by central control unit control and power supply.Single lateral flow device or several equipment can be surveyed simultaneously
If trying dry reaction (if or dry-eye disease and/or analyte), and line program is entered with different condition under independent control.Crossing current is set
Standby can be " form of n x m " dot matrix (matrix) or the form of linear array.More sides for being related to perform flow hybridization
The description of method and equipment can Ginseng see United States Patent (USP) 5,741,647, United States Patent (USP) 6,020,187 and PCT application WO/2011/
139750.Those skilled in the art can be used in United States Patent (USP) 5,741,647 or United States Patent (USP) 6,020,187
The nearly flow hybridization technology of described class, or be able to carry out any new method or equipment of flow hybridization technology to put into practice this hair
It is bright.The imaging system that hybridization device has, such as FT-Pro, can be biometrics by signal detection.Hybridization of the present invention is set
Standby can be the equipment of automation, and the biometrics function built in it can be used to carry out all analyses.Flow hybridization method is than passing
System hybridization technique has higher efficiency, the degree of accuracy and sensitivity.
Drug resistant M mycobacterium (MTB) detects
The method provided by the present invention and kit are detected to rifampin (RIF) and different cigarette with PCR and " water conservancy diversion " formula hybridization technique
Hydrazine (INH) has the Mycobacterium tuberculosis (MTB) of drug resistance.The rpoB gene mutations of MTB RNA polymerase β subunits with to sharp good fortune
Flat drug resistance is relevant；And the mutation of katG genes (catalase) and INHA genes (enoyl-ACP reductase) with to different
The resistance of cigarette hydrazine is relevant.In the present invention, specific primer be used to expand INHA, rpoB gene, and katG genes；Specificity
Primer has been found to do not have cross reaction with human genome.Primer RS-IAC serves as internal control, and it is not directed to anyone
Class or MTB genomes, for monitoring PCR amplification procedures.Probe includes ten gene mutation DNAs for being used to detect drug resistance and visited
Pin；This gene mutation has rpoB (D516V, D516G, H526D, H526Y, H526L1, S531L and S531W), katG (S315T1
And S315T2), and inhA (- 15TC/T).Probe also includes five DNA probes for being used to detect wild type MTB genomes, this
Help to verify rpoB genes, whether the pcr amplification reaction of katG genes and inhA genes has completed.Hybridized primer can be
It is biotinylated to support.
Beta Thalassemia is detected
The present invention provides the method and kit of detection betaglobulin gene mutation, and mutation includes TATA-28 (A>G),
TATA-29(A>G detection), initiation codon (G>A), codon 5 (- CT), codon 8/9 (+G), (G of codon 15>A),
Codon 16n (- C), (A of codon 17>T), (A of codon 19>G), (G of codon 26>A) (hemoglobin), codon 27/
28 (+C), codon is 30G>C, IVS1.1 (G>T), IVS1.1 (G>A), IVS1.5 (G>C), codon 41/42 (- TCTT),
(the G of codon 43>T), codon 71/72 (+A), IVS2.1 (G>A), IVS2.654 (C>T) and 619bp missing.
Hepatitis type B virus detects
The method provided by the present invention and kit are to detect 8 genotype of hepatitis B virus (genotype of hepatitis B virus
A, B, C, D, E, F, G and H) with the presence or absence of in the blood serum sample of patient Yu.
Detect sexual reverse
The method provided by the present invention and kit, to detect in each sample, such as urine, urogenital system swab (urine
Road, vagina, cervix and various lesions) and liquid based cytology sample (PreservCytTMAnd SurePathTM) protozoa, thin
Bacterium, and viral presence.Amplification control (AC) include be used for detect human DNA, with inspection result have enough cell contents and
Validity.Whether the amount of DNA that AC can be used to monitor the presence of the PCR inhibitor in PCR amplification procedures and extract is enough.There is no AC
Signal represents presence inhibitor or the amount of DNA deficiency (or in the absence of corpse or other object for laboratory examination and chemical testing) in PCR amplification procedures.Positive control (PC) includes one
Positive template nucleic acid molecules monitor the performance of PCR reagent.
DNA hybridization and round pcr can detect faster urine, urogenital system swab (urethra, vagina, cervix and
Various lesions), and the sexual reverse in liquid based cytology sample (PreservCytTM and SurePathTM).In an implementation
In scheme, methods described and the detectable 12 kinds of common venereal diseases pathogen of kit：1 kind of protozoan (trichomonas vaginalis), 7 kinds
Bacterium (chlamydia trachomatis, NEISSERIA GONORRHOEAE, mycoplasma genitalium, mycoplasma hominis, Ureaplasma urealyticum, solution sporozoite and treponemal
Body) and 4 kinds of viruses (Dan Chunpaozhenbingdu1 ＆2 types, the type of HPV 6 and 11 types).These pathogen all with cervicitis,
Urethritis, trichomoniasis are relevant with pelvic infecton.
Universal primer and pathogen specific primer that the present invention is provided, for detecting sexual reverse.Universal primer is used
To realize that the PCR of a variety of different targets simultaneously balances amplification.Universal primer sequence tests spy by PCR and gel electrophoresis
The opposite sex, to prove there is not cross reaction with other pathogens and human genome.Two universal primer sequences are added into respectively
The 5' ends of pathogen specific primer and AC specific primers.Universal primer and pathogen specific primer mark pass through PCR again
Further tested with gel electrophoresis, it was demonstrated that do not have cross reaction (Fig. 1) with other pathogen and human genome.In a reality
Apply in scheme, pathogen specific primer and two universal sequence marks (primer) are included in single PCR amplifications.In the first round
PCR, pathogen specific primer combines its specific target, and creates the pcr amplification product of a carrying universal sequence.Connect
, in the second wheel PCR, universal primer is attached to the PCR primer (Fig. 2) of universal sequence mark.
Under the PCR of optimization, we can realize balance amplification to avoid because different target pathogen DNA sequence dna has difference
Amplification efficiency and cause result to have deviation.The embodiment also assures that the amplified production of pathogen is only produced by universal primer
It is raw, with DNA probe so as to there is more accurate detection.Universal primer system described herein is equally applicable to other method and examination
Agent box detects the DR-MTB not described in the present invention, beta Globulin, hepatitis B and thrombophilia, and with it is other
Disease relevant nucleic acid or gene.
Thrombophilia is detected
The present invention provides the method for the detection gene mutation relevant with heredity thrombophilia, the gene mutation bag
Include Leiden accelerator factor Factor V Leiden (FVL) [1691G>A], factor Factor II (FII or be solidifying
Blood factor Ⅱ) [20210G>A], methylenetetrahydrofolate reductase Methylenetetrahydrofolate Reductase
(MTHFR)[677C>T] and methyl tetrahydrofolate reductase Methylenetetrahydrofolate Reductase (MTHFR)
[1298A>C]。
Although the checking data experiment of the present invention is using PCR with extension increasing sequence, any Ke Yi Productivity give birth to enough specific mesh of deal
Standard sequences can also be used with carrying out the method that identification and flow hybridization are analyzed.If resulting mesh Standard sequences deal is enough,
It need not may be expanded using PCR.Detection can be realized by detecting the mark on mesh Standard DNA or binding element.
Brief description of the drawings
Fig. 1 a show the electrophoretogram that clinical sample PCR reactants are expanded using universal primer (sequence number 285 and 286)
Picture.Only observed in the positive (i.e. CT- is positive, and NG- is positive, and HPV-6 and HPV-11 are positive) for having pathogen specific
Frequency band, this shows that Hui does not have reaction to universal primer with human DNA.Fig. 1 b, which are shown, uses pathogen specific primer (sequence number
The electrophoretic image of the PCR reactants of Human clinical's sample 263-282) is expanded with universal primer (sequence number 285 and 286).Each
Sample observation shows that the corresponding pathogen of primer has high degree of specificity to the special frequency band of correspondence special pathogen.It is similar
Situation single and multiple PCR reaction in can observe.
Fig. 2 shows the embodiment of a universal primer being used in single pcr amplification reaction and gene-specific primer.
Fig. 3 shows that the signal location of the DR-MTB detections of a DR-MTB case (left figure) and an embodiment is (right
Figure) (ruling way only for instructions).IAC internal controls monitor PCR amplification procedure.HC is hybridization control, monitors 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 way only for instructions).
Fig. 6 a show an example for explaining different β-thalassemia genotype.Fig. 6 b show using β-
The test result of the various clinical samples of thalassemia case.
Fig. 7 shows one embodiment of hepatitis type B virus test kit detection, there is 8 hepatitis type B virus bases
Because of type (A, B, C, D, E, F, G and the H of hepatitis type B virus) presence.In universal primer IAC and HC are also included within.
Fig. 8 shows that an example goes to explain different genotype of hepatitis B virus.
Fig. 9 a show the comparison of the present invention and the sensitiveness of other one venereal disease kits of manufactory.Sheet shown in Fig. 9 b
The clinical sample of the venereal disease kit of the performance of invention and other manufacturers compares.
The embodiment that Figure 10 a show a kind of venereal disease reagent array, case form and signal location.The chip can be with
Detect the presence of 12 kinds of common causative substances：1 kind of protozoan (trichomonas vaginalis), 7 kinds of bacterium (chlamydia trachomatis, gonococcus, branch
Substance, mycoplasma hominis, Ureaplasma urealyticum solves urea sporozoite, microspironema pallidum) and 4 kinds of virus (Dan Chunpaozhenbingdu1 ＆2 types,
The type of HPV 6 and 11 types).Amplification control (AC) is used to monitor the presence for having inhibitor in PCR amplification procedures and carried
Whether the amount of DNA taken is enough.Amplification control signal, which is not present, represents to have in PCR amplifications inhibitor or amount of DNA are not enough (or not deposit
In a corpse or other object for laboratory examination and chemical testing).Microspironema pallidum (TP), can be provided as positive control (PC), and its positive template nucleic acid molecules contained is monitored
The performance of PCR reagent.Figure 10 b show an example of venereal disease kit array.
Figure 11 shows the embodiment of a thrombophilia kit array.
Figure 12 shows the embodiment of a thrombophilia kit array, and its test result annotation.
The content of the invention
Following term is used to describe the present invention.Specific definitions are not illustrated such as, and the term for describing the present invention should be to
Give its ordinary meaning understood by those of ordinary skill.
As described herein, " equipotential specific oligonucleotide probe-reverse dot blot hybridization (ASO-RDB) " refers to that use can lead to
Cross the detection of the ASO-RDB target molecules of hybridizing method capture in solids.
Term " flow hybridization " as used herein refers to utilizing the technology described in U.S. Patent number 5,741,647
Crossover process.
Term " flow hybridization equipment " or " guiding device " as used herein are referred in U.S. Patent number 6,020,187
Described in equipment and/or lateral fluidic device described in PCT Application No. WO/2011/139,750 or then design
Any guiding device.
Detect pathogen and target dna
Method, primer, probe and the kit that the present invention is provided are used to detect various nucleic acid, mutation, material and cause of disease
Body/or disease.In one embodiment, the present invention is applied to detection tuberculosis, hepatitis B, hepatitis C, SARS, breathing
Road infection virus, sexual reverse, beta-globin, thrombophilia and/or presence (independent one kind or many about nucleic acid
Kind).The PCR of methods described produces amplified production, the amplified production and oligonucleotide probe hybridization using specific primer.
Gained hybridization spectrum will reflect the presence of nucleic acid, gene mutation, material, pathogen and/or disease.
In one embodiment, the hybridization of amplified production and oligonucleotide probe is using method of river diversion, horizontal water conservancy diversion side
Method or reverse guide method.In one embodiment, the oligonucleotide probe for hybridization is fixed in film.In water conservancy diversion
In the embodiment of method, the sensitiveness of hybridization depends on including the gross area of the film of the probe array.In horizontal water conservancy diversion side
In the embodiment of method, the sensitiveness of hybridization depends on including the probe across the total cross-sectional area of the film on flow direction.
In one embodiment, the invention provides a kind of side for being used to detect the presence of nucleic acid and/or allogenic material
Method, comprises the following steps：(a) nucleic acid-templated nucleic acid molecules of the amplification in Samples subjects, wherein primer are selected from sequence number
For：174-181,201-205,241-244,263-286 and 299-306, to produce individually one or more amplified productions；With
(b) amplified production and oligonucleotide probe hybridization, probe include being selected from sequence number 182-200,206-240,245-260,
287-298, and 307-314, hybridization spectrum is produced with independent one or more oligonucleotide probes, show target nucleic acid and/or
Allogenic material whether there is in Samples subjects.In one embodiment, the quantity of selected primer is 2-49；Select spy
The number of pin is 2-92.In another embodiment, selected primer quantity be 2-24；The number of selected probe be 2-
35。
In another embodiment, the invention provides a kind of method be used for detect in sample with the presence or absence of nucleic acid and/
Or allogenic material, it the described method comprises the following steps：(a) template nucleic acid molecule of the amplification in Samples subjects, wherein institute
Serial No. is selected from primer：174-181,201-205,241-244,263-286 and 299-306, it is enough independent to produce
One or more amplified productions；The amplified production and oligonucleotide probe hybridization, probe includes be selected from sequence number 182- (b)
200,206-240,245-260,287-298, and 307-314, hybridization is produced with independent one or more oligonucleotide probes
Spectrum, shows that target nucleic acid and/or allogenic material whether there is in Samples subjects.
In one embodiment, it is used to detect multi-drug resistant mycobacterium tuberculosis (DR-MTB) the invention provides one kind
Or the method for its nucleic acid, methods described comprises the steps of：(a) sample for including template nucleic acid molecule is obtained；(b) use and be selected from
Primer sequence number expands the template nucleic acid molecule for 174-181 primer, so as to obtain amplified production；By gained (c)
Amplified production and the oligonucleotide probe hybridization selected from Serial No. 182-200, gained results of hybridization show multi-drug resistant knot
Pyrenomycetes (DR-MTB) or its nucleic acid whether there is.In one embodiment, it is used to detect multidrug resistant the invention provides one kind
The method of property mycobacterium tuberculosis (DR-MTB) or its nucleic acid, methods described is comprised the steps of：(a) obtain and include template nucleic acid
The sample of molecule；(b) template nucleic acid molecule is expanded using Serial No. 174-181 primer, so as to obtain amplified production；
By the amplified production of gained and Serial No. 182-200 oligonucleotide probe hybridization, gained results of hybridization show multiple (c)
Drug resistant M bacterium (DR-MTB) or its nucleic acid whether there is.In one embodiment, primer produces signal comprising one
Label.In one embodiment, the amplified production is by method of river diversion, horizontal method of river diversion or reverse guide method and/or
About equipment and multiple oligonucleotide probe hybridizations.
In one embodiment, the above method can detect the presence (DR-MTB) of multi-drug resistant mycobacterium tuberculosis,
Or the nucleic acid being mutated with one or more of DR-MTB：One of seven mutation of rpoB genes；Two mutation of katG genes
One of；With a mutation of inhA genes.Present invention also offers a kind of kit, for detecting multi-drug resistant tuberculosis branch
The presence (DR-MTB) of bacillus or its nucleic acid, kit, which is included, to be selected from by Serial No. 182-200 primer and selected from sequence number
For 174-181 oligonucleotide probe.In one embodiment, kit is used for the presence for detecting multi-drug resistant mycobacteria
(DR-MTB), or DR-MTB nucleic acid, kit comprising Serial No. 174-181 primer and Serial No. 182-200 widow
Nucleotide probe.
Present invention also offers method of the detection with the mutation of the subject of β-thalassemia its betaglobulin, comprising
Following steps：(a) sample comprising template nucleic acid molecule is obtained from subject；(b) expanded using Serial No. 201-205 primer
Increase the template nucleic acid molecule, so as to obtain amplified production；By the amplified production of gained be selected from Serial No. 206- (c)
240 oligonucleotide probe hybridization, gained results of hybridization shows the genotype of β-thalassemia.In one embodiment,
Method of the detection with its betaglobulin gene mutation of the subject of β-thalassemia is comprised the steps of：(a) from tested
Person obtains the sample for including template nucleic acid molecule；(b) template nucleic acid point is expanded using Serial No. 201-205 primer
Son, so as to obtain amplified production；By the amplified production of gained and Serial No. 206-240 oligonucleotide probe hybridization (c),
Gained results of hybridization shows the genotype of β-thalassemia.In one embodiment, primer produces signal comprising one
Label.One implement disease in, the amplified production is by method of river diversion, horizontal method of river diversion, or reverse guide method and/or
About equipment and multiple oligonucleotide probe hybridizations.In one embodiment, the method for above-mentioned detection betaglobulin mutation can be with
Detect one of mutation of 21 betaglobulins.
The present invention also provides the kit for detecting betaglobulin gene mutation, and kit, which is included, is selected from Serial No.
201-205 primer and the oligonucleotide probe selected from Serial No. 206-240.In one embodiment, for detecting β-ball
Kit 201-205 containing Serial No. of the mutation of albumen primer and Serial No. 206-240 oligonucleotide probe.
It is used to detect hepatitis type B virus or the method for its nucleic acid present invention also offers a kind of, step is included：(a) obtain
Sample comprising template nucleic acid molecule；(b) template nucleic acid molecule is expanded using the primer selected from Serial No. 241-244,
So as to obtain the amplified production of hepatitis type B virus；By the amplified production of gained and widow selected from Serial No. 245-260 (c)
Nucleotide probe hybridizes, and gained results of hybridization shows that hepatitis type B virus or its nucleic acid whether there is.In one embodiment, use
Comprised the steps of in the method for detection hepatitis type B virus or its nucleic acid：(a) sample for including template nucleic acid molecule is obtained；
(b) template nucleic acid molecule is expanded using Serial No. 241-244 primer, so as to obtain amplified production；By gained (c)
Amplified production and Serial No. 245-260 oligonucleotide probe hybridization, gained results of hybridization show hepatitis type B virus or
Its nucleic acid whether there is.In one embodiment, primer includes the label of a generation signal.In one embodiment, it is described
Amplified production horizontal method of river diversion or reverse guide method and/or is visited by method of river diversion about equipment and multiple oligonucleotides
Pin hybridizes.
In one embodiment, the above method can detect hepatitis type B virus or its nucleic acid, the hepatitis B
Poison is selected from genotype A to H.It is used to detect hepatitis type B virus or the kit of its nucleic acid present invention also offers a kind of, comprising
The oligonucleotide probe of primer and Serial No. 245-260 selected from Serial No. 241-244.In one embodiment, it is used for
Detect primer of the kit comprising Serial No. 241-244 and Serial No. 245-260 of hepatitis type B virus or its nucleic acid
Oligonucleotide probe.
It is described for detecting that subject whether there is sexual reverse or its nucleic acid present invention also offers a kind of method
Method is comprised the steps of：(a) sample comprising template nucleic acid molecule is obtained from the subject；(b) using selected from sequence number
The template nucleic acid molecule is expanded for 263-286 primer, so as to obtain amplified production；(c) by the amplified production of gained with
Oligonucleotide probe hybridization selected from Serial No. 287-298, gained results of hybridization shows that sample whether there is Come certainly or can draw
Play the nucleic acid of the pathogen of venereal disease.In one embodiment, the invention provides a kind of method, for detecting whether subject deposits
In sexual reverse or its nucleic acid, comprise the steps of：(a) sample comprising template nucleic acid molecule is obtained from the subject；
(b) template nucleic acid molecule is expanded using Serial No. 263-286 primer, so as to obtain amplified production；By gained (c)
Amplified production and Serial No. 287-298 oligonucleotide probe hybridization, gained results of hybridization shows that sample whether there is Come
From or can cause venereal disease pathogen nucleic acid.In one embodiment, primer includes the label of a generation signal.At one
In embodiment, the amplified production is by method of river diversion, horizontal method of river diversion or reverse guide method and/or about equipment and many
Individual oligonucleotide probe hybridization.
In one embodiment, for detecting that the method for sexual reverse in subject can detect following venereal disease cause of disease
Body:Trichomonas vaginalis, chlamydia trachomatis, NEISSERIA GONORRHOEAE, mycoplasma genitalium, mycoplasma hominis, Ureaplasma urealyticum solves spore
Worm, microspironema pallidum, herpes simplex virus type 1, herpes simplex virus type 2, the type of HPV 6, and HPV
11 type are described present invention also offers a kind of kit for being used to detect the presence of sexual reverse or its nucleic acid in subject
Kit includes the primer selected from Serial No. 263-286 and the oligonucleotide probe selected from Serial No. 287-298.At one
In embodiment, for detecting that the kit of sexual reverse or its nucleic acid in Samples subjects includes Serial No. 263-286's
The oligonucleotide probe of primer and Serial No. 287-298.
The invention provides the method for detecting the gene mutation relevant with thrombophilia, methods described comprising with
Lower step：(a) sample for including template nucleic acid molecule is obtained；(b) it is described using the primer amplification selected from Serial No. 299-306
Template nucleic acid molecule, so as to obtain amplified production；By the amplified production of gained and widow selected from Serial No. 307-314 (c)
Nucleotide probe hybridizes, and gained results of hybridization shows that the gene mutation relevant with thrombophilia whether there is.In a reality
Apply in example, the primer includes the label of a generation signal.In another embodiment, methods described is used to detect and thrombus
The relevant gene mutation of formability, methods described is comprised the steps of：(a) sample for including template nucleic acid molecule is obtained；(b)
The template nucleic acid molecule is expanded using Serial No. 299-306 primer, so as to obtain amplified production；By gained (c)
The oligonucleotide probe hybridization of amplified production and Serial No. 307-314, gained results of hybridization, which is shown with thrombophilia, to be had
The gene mutation of pass whether there is.In one embodiment, the primer includes the label of a generation signal.In an implementation
In example, the amplified production is by method of river diversion, horizontal method of river diversion, reverse guide method and/or about equipment and multiple widows
Nucleotide probe hybridizes.
In one embodiment, methods described is used to detect the gene mutation relevant with thrombophilia, the gene
Selected from Leiden accelerator factor FactorV Leiden (FVL), factor FactorII (FII or factor II),
With methylenetetrahydrofolate reductase Methylenetetrahydrofolate Reductase (MTHFR).The present invention is also provided
It is used for the kit for detecting the gene mutation relevant with thrombophilia, the kit is included selected from Serial No. 299-
306 primer and the oligonucleotide probe selected from Serial No. 307-314.In one embodiment, the kit includes sequence
Row number is 299-306 primer and Serial No. 307-314 oligonucleotide probe.
By quoting, following experimental detail, may be better understood the present invention.However, it will be understood by those skilled in the art that
The embodiment provided is only as illustration, rather than Xian System the scope of the present invention.The scope of the present invention is by by subsequent right
It is required that being defined.
In this application, different bibliography or publication be refer to.These references or the full text and public affairs of publication
Open and be incorporated into the application, so that the situation of the present invention is described more fully with.It should be pointed out that Transitional Language "comprising" with '
Including ' ﹑ ' containing ' or ' with ... for characteristic ' be synonymous, be it is inclusive or open, it is central to be not precluded from not arranging in addition
The element or method and step of act.
Embodiment 1
Test program
For DNA, PCR amplification, hybridization, detection sample and interpretation of result separating step, such as WO/2011/139750 or
It is known as those skilled in the art and may apply to the present invention in relevant program and technology.Various internal contrasts (IC) can wrap
Include in the testing experiment, with validating DNA sample, PCR amplifications and result colour developing.The size and form of each water conservancy diversion membrane array,
It is maximized cost-effective to reach the need for according to individual, or for optimizing the use of reative cell.
Embodiment 2
Genotyping multi-drug resistance tuberculosis
In one embodiment, the invention provides the method for DR-MTB Genotypings and kit.Methods described with
Kit uses polymerase chain reaction (PCR) and " water conservancy diversion " hybridization technique, for detecting to rifampin (RIF) and isoniazid
(INH) there is the Mycobacterium tuberculosis (MTB) of drug resistance.For expanding rpoB genes, katG genes and the corresponding of INHA genes are drawn
Thing has been found to do 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 procedures.The gene mutation of probe in detecting drug resistance used include rpoB genes (D516V,
D516G, H526D, H526Y, H526L1, S531L and S531W), katG genes (S315T1 and S315T2), and inhA genes (-
15TC/T).Also there are five probes to be used to detect wild type MTB mutation, this helps to verify rpoB genes, katG genes and
Whether the pcr amplification reaction of inhA genes has completed.In one embodiment, primer is biotinylated to be hybridized.
In one embodiment, the invention provides the side for detecting multiple-drug resistance tuberculosis mycobacteria (DR-MTB)
Method, comprises the following steps：(a) sample for including template nucleic acid molecule is obtained；(b) with being selected from Serial No.：174-181's draws
Thing, expands the template nucleic acid molecule, so as to produce amplified production；Amplified production be selected from Serial No. (c)：182-200
Oligonucleotide probe constituted group hybridization, gained hybridization spectrum will show multiple-drug resistance tuberculosis mycobacteria presence.One
In individual embodiment, the present invention can detect multi-drug resistant mycobacterium tuberculosis, and with it is following wherein one or more dash forward
The presence of change：Seven mutation in rpoB genes；Two mutation in katG genes；A mutation in inhA genes.One
In individual embodiment, amplified production is the horizontal method of river diversion by method of river diversion, or reverse guide method, with multiple few nucleosides
The hybridization of acid probe.
Fig. 3 shows that each position of a DR-MTB case (left figure) and probe and signal (right figure) is embodied.IAC is interior
Portion's amplification control, to monitor PCR amplification procedures.HC is hybridization control, monitors crossover process.Fig. 4 shows that one is used to detect
The embodiment of DR-MTB array configurations.In one embodiment, there are the MTB strain quilts of drug resistance to RIF and isoniazid respectively
Detect.In another embodiment, the MTB bacterial strains for having drug resistance (multi-drug resistant) to RIF and INH are detected
Come.In one embodiment, neither resistance to rifampin (RIF), nor the MTB bacterial strains of resistance to isoniazid (INH) are detected
Come.Detection includes various controls.
Primer
| Primer I D | Primer sequence (5'-3') | 5' is modified | Sequence number |
| MTB-DR-Rpob-F | TCAACATCCGGCCGGTGGTC | Biotin | 174 |
| MTB-DR-Rpob-R | CCGGCACGCTCACGTGACAGA | Biotin | 175 |
| MTB-DR-KatG-F | TGGCACCGGAACCGGTAAGGA | Biotin | 176 |
| MTB-DR-KatG-R | CGCCAGCAGGGCTCTTCGT | 177 | |
| MTB-DR-inhA-F | AAGTTCCCGCCGGAAATCGCAG | Biotin | 178 |
| MTB-DR-inhA-R | CCGGTAACCAGGACTGAACGGGAT | 179 | |
| RS-IAC-F | CGAGTTCCCGCCCATAACC | 180 | |
| RS-IAC-R | GGAAGTTGCAACGCCGTCC | Biotin | 181 |
Probe
Here is that some are used for the example of PCR and hybridization:
| Composition | Capacity (μ L) |
| PCR is pre-mixed | 19.6 |
| Primer is pre-mixed | 2 |
| IAC | 0.1 |
| Taq polymerase | 0.4 |
| Nuclease-free water | 0.9 |
| Template nucleic acid molecule | 2 |
| Amount to | 25.00 |
Hybridize one embodiment of agreement:
Adjustment equipment temperature (downward)
Adjustment equipment temperature (up-regulation)
Embodiment 3
β Mediterranean Genotyping
The invention provides a kind of detection of the betaglobulin gene mutation of system, the gene mutation includes TATA-28
(A>G), TATA-29 (A>G), initiation codon (G>A), the detection of codon 5 (- CT), codon 8/9 (+G), codon 15
(G>A), codon 16 (- C), (A of codon 17>T), (A of codon 19>G), (G of codon 26>A) (Hb E), password
Sub 27/28 (+three), codon is 30G>C, IVS1.1 (G>T), IVS1.1 (G>A), IVS1.5 (G>C), codon 41/42 (-
TCTT), (G of codon 43>T), codon 71/72 (+A), IVS2.1 (G>A), IVS2.654 (C>T) and 619bp missing.
Present invention also offers the method that betaglobulin in detection Samples subjects is mutated, methods described includes following step
Suddenly：(a) sample comprising template nucleic acid molecule is obtained from subject；(b) with being selected from Serial No.：201-205 primer institute group
Into group expand the template nucleic acid molecule, so as to produce amplified production；Amplified production be selected from Serial No. (c)：206-
The group hybridization that 240 oligonucleotide probe is constituted, gained hybridization spectrum will indicate that the genotype of β-thalassemia.In a reality
Apply in scheme, primer includes a signal and produces label.In one embodiment, amplified production is horizontal by method of river diversion
Method of river diversion, or reverse guide method, with multiple oligonucleotide probe hybridizations.In one embodiment, above method ball with
Any one of which among 21 kinds of beta-globin mutation of detection.
Fig. 5 shows the implementation of a kind of β-thalassemia case and its signal location (ruling way only for instructions in figure)
Scheme.Fig. 6 a show an example of different beta-thalassemia genotype.Fig. 6 b are shown on using β-thalassemia
The test result of the various clinical samples of case, shows that the present invention can recognize the genotype of β-thalassemia from human sample.
Primer sequence (addition AF007546.1):
Probe sequence (addition AF007546.1):
| Probe I D | Sense/antisense chain | 5' is modified | 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 | GCACCTGACTCCTGAGG | 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 that some are used for the exemplary protocols of PCR and hybridization:
| Composition | Capacity (μ L) |
| PCR is pre-mixed | 19.6 |
| Taq polymerase | 0.4 |
| Template nucleic acid molecule | 2to 5.0* |
| Nuclease-free water | It is variable |
| Amount to | 25.00 |
Hybridize one embodiment of agreement:
Data are read as the mutation of β balls:
Embodiment 4
Hepatitis B virus gene typing
Present invention also offers a kind of method for detecting hepatitis type B virus, methods described includes step：(a) obtain
Sample comprising template nucleic acid molecule；(b) with selected from Serial No.：The group that 241-244 primer is constituted expands the template
Nucleic acid molecules, so as to produce amplified production；Amplified production be selected from Serial No. (c)：245-260 oligonucleotide probe institute
The group hybridization of composition, gained hybridization spectrum will show the presence of hepatitis type B virus.In one embodiment, primer includes one
Signal produces label.In one embodiment, amplified production is by method of river diversion, horizontal method of river diversion, or reverse guide side
Method, with multiple oligonucleotide probe hybridizations.In one embodiment, general probe sequence number 257 be used to capture various bases
Because of the nucleic acid of the hepatitis type B virus of type.
Fig. 7 shows 8 genotype of hepatitis B virus (B-mode livers that the Hepatitis B Virus Reagents box of one is detected
A, B, C, D, E, F, G and the H type of scorching virus) embodiment.Fig. 8 shows one of the different genotype of hepatitis B virus of detection
Example.
Primer sequence:
Probe sequence:
Hepatitis type B virus positive control sequence [560bp] (pUC57 carriers):
TGGGATTCTATATAAGAGGGAAACTACACGTAGCGCCTCATTTTGCGGGTCACCATATTCTTGGGAACAAGAGCTAC
ATCATGGGAGGTTGGTCATCAAAACCTCGCAAAGGCATGGGGACGAACCTTTCTGTTCCCAACCCTCTGGGATTCTT
TCCCGATCATCAGTTGGACCCTGCATTCGGAGCCAATTCAAACAATCCAGATTGGGACTTCAACCCCATCAAGGACC
ACTGGCCACAAGCCAACCAGGTAGGAGTGGGAGCATTCGGGCCAGGGTTCACTCCCCCACACGGAGGTGTTTTGGGG
TGGAGCCCTCAGGCTCAGGGCATATTGGCCACAGTGCCAGCAGTGCCTCCTCCTGCCTCCACCAATCGGCAGTCAGG
AAGGCAGCCTACTCCCATCTCTCCACCTCTAAGAGACAGTCATCCTCAGGCCATGCAGTGGAATTCCACAGCTTTCC
ACCAAGCTCTGCAAGATCCCAGAGTCAGGGGCCTGTATTTTCCTGCTGGTGGCTCCAGTTCAGGAACACTCAACCCT
GTTCCAACTATTGCCTCTCAC (sequence number 261)
Internal control sequence [500bp] (pUC57 carriers):
GAGACAGGTTCGTCCAATCCCGTGCCGCGGCCTTGGCAGGGGGTTCGCAGGCCCCACCCGAAGCGTTGCTGAAGGCT
CAGGCCTCTGAGCGACAAAAGCTTTAAACGCGAGTTCCCGCCCATAACCTGGACCGAATGCGGGACCATGCATCGTT
CCACTGTGTTTGTCCCATGTAGGACGGGCGCAAGGCGTGCTTAGCTCAGCCTCGAATGCCTCGTATCATTGTGCACC
CGCCGGTCACCAGCCAACGATGTGCGGACGGCGTTGCAACTTCCGGGGCCCAACCTGACCGTCCTGGGTACCGCACT
CTGGGCAGTGCGAGGTAATGCCAGTCGCCCAGTGCCGAACAACACCTGACCTAACGGTAAGAGGCTCACATAATGGC
TCCGCCGGCGCGCCCAGGGTACATTAGGTCAGCATCGGATGGACTGACATGAACCTTCACACCGAAGCGGAAACGGG
TGCGTGGACCAGCGAGGAGCAAACGAAAATTCCTGGCC (sequence number 262)
The following is some PCR agreements and the example of hybridization agreement:
| Composition | Capacity (μ L) |
| Hepatitis type B virus primer is pre-mixed | 19.6 |
| IAC | 0.1 |
| Taq polymerase | 0.3 |
| Template nucleic acid molecule | 5.0* |
| Nuclease-free water | It is variable |
| Amount to | 25.00 |
* total hepatitis type B virus viral DNA is more than 4000IU/mL. in suggestion serum
This temperature curve is applied to Applied Biosystems, Inc.'s PerkinElmer (ABI-PE) 9600, gene magnification PCR
System 9700, Veriti (Applied Biosystems companies), PTC-200 (MJ researchs)..
Hybridize one embodiment of agreement:
Embodiment 5
Genotyping, and a variety of sexual reverses are detected simultaneously
In one embodiment, the method that the present invention provides detection sexual reverse, the pathogen includes but not office
It is limited to, protozoan, bacterium and virus.In one embodiment, sexual reverse is present in urine, and urogenital system is wiped
Sub (urethra, vagina, cervix and damage) and liquid based cytology sample (PreservCytTMAnd SurePathTM).In a reality
Apply in scheme, amplification control (AC) includes the material for being used to detect human DNA.With PCR amplification urine urogenital system swab (urine
Road, vagina, cervix and damage) and liquid based cytology sample (PreservCytTMAnd SurePathTM) the middle target DNA extracted,
Again by hybridization, reach more rapidly, it is sensitiveer and more specifically detect venereal disease.In one embodiment, methods described can be with
12 kinds of common sexual reverses of detection, including a kind of protozoan (trichomonas vaginalis), 7 kinds of bacterium (chlamydia trachomatises, gonorrhoea
Neisseria, mycoplasma genitalium, mycoplasma hominis, Ureaplasma urealyticum solves sporozoite, microspironema pallidum) and 4 kinds of virus (simple blisters
The Xing ＆2 types of exanthema virus 1, the type of HPV 6 and 11 types).These pathogen all with cervicitis, urethritis, trichomoniasis and basin
Chamber inflammation is relevant.In another embodiment, universal primer (S) and/or amplification control are included in test.
Present invention also offers a kind of method, for detecting sexual reverse present in Samples subjects, methods described
The step of include：(a) sample comprising template nucleic acid molecule is obtained from subject；(b) by selected from Serial No.:263-286
The group that is constituted of primer, the template nucleic acid molecule is expanded, so as to produce amplified production；Amplified production be selected from sequence (c)
Row number is：287-298 oligonucleotide probe hybridization, gained hybridization spectrum will be displayed with nucleic acid fragment from sexual reverse.
In one embodiment, primer includes a signal generation label.In one embodiment, with multiple oligonucleotide probes
The hybridization of amplified production be horizontal method of river diversion or reverse guide method and/or relevant equipment in a method of river diversion.
Sequence number 263-284 primer is made up of two parts：5' parts are artificial synthesized nucleotide sequences, and target cause of disease
Body is not homologous, is not also related to the sequence of any organism that be able to can be found in the mankind；3' parts are pathogen specific sequences
Row, enable primer to be effectively attached to the nucleic acid of corresponding pathogen, to produce amplified production.The 5' parts of all primer sequences are logical
Use sequence.Universal primer sequence design is by giving careful consideration and testing, can exclude the other biological body in amplified sample, example
Such as other pathogens and any DNA (Fig. 1) of human genome.Such as those are disclosed in the present invention selected
Select and be verified as specific universal sequence and then merge with various pathogen specific sequences, obtain pathogen specific primer, its energy
It is enough to produce the amplified production for including universal sequence.These amplified productions turn into second of PCR template nucleic acid molecule, then make again
Use Serial No.：285 and 286 universal primer expands all template nucleic acid molecules with same efficiency.Therefore the expansion linearly produced
Volume increase thing ultimate density can be directly proportional with primary target DNA concentration.Even if target copy number is low in sample, still can amplify with
Reach that suitable concentration detects the positive again, can correct mistake using universal primer, greatly improve sensitivity.In an embodiment
In, the PCR of two steps reacts to be carried out in single test tube；Pathogen specific primer therein passes through its gene specific 3' areas
Domain is attached to target molecule, to produce the primary amplification product containing 5' universal sequences.These amplified productions are used as described second
The template nucleic acid molecule of group primer (universal primer), to produce the amplified production containing signal and be detected by hybridization.
Fig. 2 shows the embodiment of an amplification scheme.
Because the present invention uses universal primer, thus it is more accurate than the detection method of in the market other sexual reverse kits
Really, it is sensitiveer.Just as illustrated in fig. 9, the present invention can detect as little as 50-300 sexual reverse copy number, and another is manufactured
The detection of the kit of factory will at least have 500 sexual reverse copy numbers；Therefore the present invention can detect relatively low pathogen
The sample of concentration.
Fig. 9 b show the performance comparision of other venereal disease check reagent boxes of the present invention and in the market.As a result quantitative PCR is passed through
(qPCR) four tests and actual gene type are confirmed.After the genotype for confirming pathogen, the present invention (3 and 4 are tested in such as figure) ratio
Other kits are less to there is error result (1 and 2 are tested in such as figure).In addition, detectable multiple pathogens (such as Fig. 9 b of the present invention
It is shown), therefore, compared with the market other kits, 12 kinds of pathogen of detection are highest number to the present invention simultaneously.Infection
The incidence of disease after HPV6 and HPV11 is very high, therefore covering of the present invention to HPV6 types and 11 types is highly useful.Genital wart
It is a kind of venereal disease with hyperinfection power, wherein 90% case is due to caused by infection HPV6 and HPV11.
Figure 10 a show the embodiment of a detection venereal disease array, and the detectable 12 kinds of common venereal diseases pathogen of array are deposited
：1 kind of protozoan (trichomonas vaginalis), 7 kinds of bacterium (chlamydia trachomatis, NEISSERIA GONORRHOEAE, mycoplasma genitalium, mycoplasma hominis,
Ureaplasma urealyticum, solves sporozoite, microspironema pallidum) and 4 kinds of viruses (herpes simplex virus type 1 and 2 types, the type of HPV 6
With 11 types).Amplification control (AC) shows whether result is effective.Figure 10 b show the example of an explanation venereal disease array.At one
In embodiment, protozoan, bacterium and virus is measured separately.In another embodiment, protozoan, bacterium and disease
Malicious (multiple infection) is detected simultaneously.
In one embodiment, the template nucleic acid molecule of microspironema pallidum (TP) be provided as positive control (PC) with
In the performance of monitoring PCR reagent.If occurring without TP positive control (PC) signal in a test, this is probably because described
DNA is contaminated and degenerated because of long-term storage or nuclease.If however, the positive signal that test sample is produced is any disease
Substance, testing result is still effective.In positive control (PC) test, because positive control sample only includes microspironema pallidum
(TP), but without endogenic human gene group DNA, it will cause to lack AC signals.In the presence (PC) of the AC signals of positive control
Test show control test be it is invalid, this be probably due to pollution and experiment answer the repetition.
Such as AC signals are occurred without, and this amount of DNA represented in test sample is not enough or is not present.If however, test sample
The positive signal of any pathogen is produced, test is still effective.Due to that may have the competition from other pathogens DNA,
AC signals may be occurred without in some samples.In this case, as long as there is one or more positive signals on array, detection knot
Fruit is still effective.
In another embodiment, the array box and/or device used during water conservancy diversion has different form and chi
It is very little., also can again plus array point is for detection such as extra cause of disease outside array using 10 points, it is also possible to 35 lattice arrays
The gene mutation of body gene hypotype and/or antibody-resistant bacterium.Detect that signal also can be by using the shadow with FT-Pro hybridization devices
As system to reach digitlization.In another embodiment, hybrid device used is an automation equipment, there is binding analysis
With digitizing function.
Present invention detection method more existing than in the market and kit are more convenient, because the present invention can be expanded and detect simultaneously
Multiple targets, with high sensitivity and cover 12 kinds of sexual reverses, including are excluded by the most of available reagent box of in the market
HPV subgenotypes outside.By coupling with flow hybridization, the present invention allows rapid sensitive and high-throughout sexual reverse to examine
Survey.
Primer sequence:
Probe sequence:
| Cause of disease/target | Probe | Probe sequence (5 ' to 3 ') | 5' is modified | 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 examples PCR and hybridization agreement:
| Composition | Capacity (μ L) |
| PCR is pre-mixed | 18.6 |
| Venereal disease primer is pre-mixed | 1.0 |
| DNA Taq polymerases | 0.4 |
| Venereal disease DNA positive controls | Most 5.0* |
| Nuclease-free water | It is variable |
| Amount to | 25.0 |
* the scope of the suggestion of STb gene:5ng–100ng.
Above thermocycling program is to be suitable forThermal Cycler (Life technologies) and
S1000TMThermal Cycler (Bio-rad) more than thermocycling programs can be modified rear for other thermal cycler (slopes
Rate is up to 3 DEG C/sec).
Hybridize one embodiment of agreement:
Embodiment 6
The relevant Genotyping of thrombophilia
Present invention also offers the method for detecting the gene mutation relevant with thrombophilia, methods described is included
Following steps：(a) sample for including template nucleic acid molecule is obtained；(b) using the primer amplification institute selected from Serial No. 299-306
Template nucleic acid molecule is stated, so as to obtain amplified production；By the amplified production of gained be selected from Serial No. 307-314 (c)
Oligonucleotide probe hybridization, gained results of hybridization shows that the gene mutation relevant with thrombophilia whether there is.At one
In embodiment, the primer includes the label of a generation signal.In one embodiment, the amplified production passes through water conservancy diversion side
Method, horizontal method of river diversion or reverse guide method and multiple oligonucleotide probe hybridizations.In one embodiment, the above method can
Leiden accelerator factor Factor V Leiden (FVL), factor FactorII (FII or blood coagulation second are selected to detect
The factor), and methylenetetrahydrofolate reductase Methylenetetrahydrofolate Reductase (MTHFR) gene
Mutation.
Figure 11 shows the embodiment of a thrombophilia kit array.Figure 12 shows that thrombophilia is tried
One annotation example of its test result of agent box.
Primer sequence:
| Primer | Primer sequence (5'-3') | 5' is modified | Sequence number |
| FVL_F | GAAAATGATGCCCAGTGCTT | / | 299 |
| FVL_R | TTGAAGGAAATGCCCCATTA | Biotin | 300 |
| FII_F | GAACCAATCCCGTGAAAGAA | / | 301 |
| FII_R | AGCTGCCCATGAATAGCACT | Biotin | 302 |
| MTHFR677_F | GGTTACCCCAAAGGCCACC | Biotin | 303 |
| MTHFR677_R | AAGCGGAAGAATGTGTCAGC | Biotin | 304 |
| MTHFR1298_F | TTTGGGGAGCTGAAGGACTA | / | 305 |
| MTHFR1298_R | CTTTGTGACCATTCCGGTTT | Biotin | 306 |
Probe sequence:
Reaction mixture:
| Composition | Capacity (μ L) |
| Thrombophilia PCR are pre-mixed | 21.6 |
| Thrombophilia primers are pre-mixed | 1.0 |
| Enzyme mixation | 0.4 |
| Template nucleic acid molecule | Most 2.0 |
| Nuclease-free water | It is variable |
| Amount to | 25.00 |
* the scope of the suggestion of DNA total amounts：10ng–100ng.
One embodiment of hybridization procedures:
Claims (10)
1. a kind of kit for being used to detect the gene mutation relevant with thrombophilia, it is characterised in that include sequence number
The oligonucleotide probe of primer and Serial No. 307-314 for 299-306.
2. kit according to claim 1, it is characterised in that wherein described primer includes the label for producing signal.
3. kit according to claim 1, it is characterised in that the kit can detect one or more selected from Leiden
The gene mutation of accelerator factor, factor and methylenetetrahydrofolate reductase.
4. kit according to claim 1, it is characterised in that the kit can detect one or more selected from following
Gene mutation：
Leiden accelerator factor [1691G>A]；
Factor [20210G>A]；
Methylenetetrahydrofolate reductase [677C>T]；With
Methylenetetrahydrofolate reductase [1298A>C].
5. any kit according to claim 1-4, it is characterised in that the kit coordinates method of river diversion, horizontal stroke
Hybridized to method of river diversion or reverse guide method.
6. Serial No. 299-306 primer and Serial No. 307-314 oligonucleotide probe are being prepared for detection and blood
Purposes in the kit of the method for the relevant nucleic acid of bolt formability, it is characterised in that methods described is comprised the steps of：
(a) sample containing template nucleic acid molecule is obtained；
(b) template nucleic acid molecule is expanded using Serial No. 299-306 primer, so as to obtain amplified production；With
(c) by the amplified production of gained and Serial No. 307-314 oligonucleotide probe hybridization, gained results of hybridization show and
The relevant nucleic acid of thrombophilia whether there is.
7. purposes according to claim 6, it is characterised in that wherein described primer includes the label for producing signal.
8. purposes according to claim 6, it is characterised in that this method can detect one or more selected from Leiden blood coagulation
The gene mutation of accelerator factor, factor and methylenetetrahydrofolate reductase.
9. purposes according to claim 6, it is characterised in that this method can detect one or more selected from following base
Because of mutation：
Leiden accelerator factor [1691G>A]；
Factor [20210G>A]；
Methylenetetrahydrofolate reductase [677C>T]；With
Methylenetetrahydrofolate reductase [1298A>C].
10. any purposes according to claim 6-9, it is characterised in that wherein described amplified production passes through water conservancy diversion
Method, horizontal method of river diversion or reverse guide method and multiple oligonucleotide probe hybridizations.
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| CN201611054755.5A CN106906306B (en) | 2013-03-15 | 2014-03-17 | Rapid and sensitive genotype identification and nucleic acid detection |
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| US201361791933P | 2013-03-15 | 2013-03-15 | |
| US61/791,933 | 2013-03-15 | ||
| PCT/CN2014/000275 WO2014139330A1 (en) | 2013-03-15 | 2014-03-17 | Rapid genotyping analysis and kits thereof |
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| ES2752761T3 (en) | 2014-07-24 | 2020-04-06 | Abbott Molecular Inc | Compositions for the detection and analysis of mycobacterium tuberculosis |
| CN116042880A (en) * | 2014-10-10 | 2023-05-02 | 新泽西鲁特格斯州立大学 | Polymerase chain reaction primer and probe of 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 |
| US10526664B2 (en) | 2015-07-14 | 2020-01-07 | Abbott Molecular Inc. | Compositions and methods for identifying drug resistant tuberculosis |
| CN112135914A (en) * | 2018-01-09 | 2020-12-25 | 雅培分子公司 | Assay for the detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis and Mycoplasma genitalium |
| WO2019152657A1 (en) * | 2018-02-03 | 2019-08-08 | Simple Healthkit, Inc. | Reliable, comprehensive, and rapid sexual health assessment |
| WO2019154996A1 (en) * | 2018-02-09 | 2019-08-15 | Genincode Uk, Ltd. | Prediction of pregnancy loss |
| 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 |
| 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 |
| 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 |
| 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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| US6043035A (en) * | 1997-11-03 | 2000-03-28 | Rijks University Leiden | Method for determining a risk factor for thrombosis |
| US7732138B2 (en) * | 2001-11-07 | 2010-06-08 | Diagcor Bioscience Incorporation Limited | Rapid genotyping analysis and the device thereof |
| US20110111389A1 (en) * | 2001-11-07 | 2011-05-12 | Diagcor Bioscience Incorporation Limited | Rapid genotyping analysis for human papillomavirus and the device thereof |
| EP1704253A1 (en) * | 2004-01-15 | 2006-09-27 | THE GOVERNMENT OF THE UNITED STATES OF AMERICA, as represented by THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES | Method evolved for recognition of thrombophilia (mert) |
| CN101591700A (en) * | 2008-05-29 | 2009-12-02 | 北京华安佛医药研究中心有限公司 | A kind of test kit, method and purposes of predicting the thrombosis disease occurrence risk |
| WO2010058357A1 (en) * | 2008-11-19 | 2010-05-27 | Diagcor Bioscience Incorporation Ltd. | Nucleotide sequences, methods and kits for detecting hpv |
| CN101864479A (en) * | 2009-04-17 | 2010-10-20 | 吴奇涵 | Application, detection method and kit of MTHFR (Methylene Tetrahydrofolate Reductase) gene SNP (Single Nucleotide Polymorphism) guide folic acid and related nutrient element intake |
| CN102732600A (en) * | 2011-04-13 | 2012-10-17 | 上海芯超生物科技有限公司 | Genetic evaluation and suit method for individualized tumor therapy |
| CN102851365A (en) * | 2012-08-31 | 2013-01-02 | 唐爱发 | Primers, kit and method for identification of polymorphism of C677T of human MTHFR gene |
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| CN105392896A (en) | 2016-03-09 |
| CN106906306A (en) | 2017-06-30 |
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