CN1896272B - Method for inspecting infectious gene MBL related to SARS CoV infection and MBL infectious gene - Google Patents
Method for inspecting infectious gene MBL related to SARS CoV infection and MBL infectious gene Download PDFInfo
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Abstract
The present invention relates to the PCR product sequencing assay of mannose-binding lectin gene (MBL) correlative with the susceptibility of SARS-CoV infection. The present invention also relates to the assay of the haplotypes correlative with the susceptibility of SARS-CoV infection that is composed of three single nucleotide polymorphism sites(SNPs): MBL gene G-550C,G-221C and G+230A. The said assay is to determine the genotype of the three SNPs by sequencing the PCR product first and then to calculate the haplotypes with PHASE programme. The present invention further relates to the detection kit of the above SNPs and the usage. Furthermore, the present invention combines the G+230A polymorphism sites and the haplotype composed of G-550C,G-221C and G+230 with SARS-CoV susceptibility, confirms that MBL is a new susceptibility gene included in SARS-CoV infection, and provides new genetic counseling content for the prevention and control of SARS-CoV infection of the population.
Description
Technical field
The present invention relates to SARS (Severe Acute Respiratory Syndrome) (Severe Acute Respiratory Syndrome, SARS) individual susceptibility and the genetics detection method thereof in crowd's prevention.Particularly, the present invention relates to and sars coronavirus (SARSCoronavirus, SARS-CoV) relevant tumor susceptibility gene mannose binding lectin (mannose-binding lectin, MBL) the gene genotype detection method of infection.The invention further relates to the genotypic detection kit of above-mentioned tumor susceptibility gene, and the application of this test kit.In addition, the invention still further relates to above-mentioned detection method and tumor susceptibility gene in the prevention of SARS and the application in control and the genetic counseling.
Background technology
SARS is the human communicable disease of determining recently, is caused by a kind of novel coronavirus SARS-CoV.By the end of in by the end of June, 2003, short half a year, the case of SARS more than 8000 was reported, wherein 700 many cases death altogether in 25 many in the interior whole world of time more than the country.The bamboo telegraph of SARS, strong clinical progress and high mortality infectious, that be difficult to expect make it become the interior serious threat of global range.Given this, effectively prevent and treat the important goal that this disease is China and even world's public health.
Data shows that individual susceptibility for SARS is different; Such as, the seropositive individuality of SARS-CoV may be suffered from SARS, also may only show slight infection symptoms.As other most of communicable diseases, the development of SARS may also be the result of complex interactions between pathogenic micro-organism, host genetic factor and the environment.That previous existing report shows is old, diabetes and heart trouble are the risk factors of SARS development; Yet the body inherited genetic factors is still understood seldom for the influence degree of SARS susceptibility.Though existing report shows that the polymorphism (as HLA-B*4601, HLA-B*0703 and HLA-DRB1*0301) of human mhc (HLA) gene is relevant with the risk that SARS takes place; yet we know that the acquired immune response needs the regular hour from identification pathogenic micro-organism (as SARS-CoV) to producing the abundant specific antibody with provide protection.Therefore, during the acquired immune response can't provide effective provide protection to body, early stage nonspecific inherent immunity reaction probably play a part aspect the susceptibility of decision disease even more important.
MBL is a kind of serum protein that belongs to collectin (collectin) family, undertakes the key player in inborn inherent immunity reaction.MBL is the acute phase reactive protein in a kind of liver cell source, and it can be attached on the sugared structural domain of the repetition seminose of bacterium, fungi, virus and falciparum surface of invasion body and N-acetylglucosamine by its a plurality of lectin structural domains.These positions of MBL bonded are characteristic structures of pathogenic micro-organism, do not exist on the surface of mammalian cell.After on being attached to pathogenic micro-organism, MBL can cause two kinds of provide protections at least.The first, MBL is by the activation of lectin pathway mediation complement system, and this does not need the participation of antibody; The second, MBL can directly impel by the collectin acceptor and engulfs opsonization.Exsomatize, show all that in the body experiment MBL defective may have material impact to the activation of inherent immunity system, and the risk that makes body be subjected to severe infections increases.
Known, on the S of the SARS-CoV that virus infection is played an important role albumen, have the discernible sugared structural domain types of MBL such as seminose.People find also that in chicken MBL can take place in the participation before and infectious bronchitis coronavirus (IBV) in humoral immune reaction.Because IBV is the same with SARS-CoV to be the member of coronavirus family, therefore, might also can be attached on the S albumen of SARS-CoV by MBL, and may bring into play its antivirus action by two kinds of approach above-mentioned.
The content of MBL in serum is controlled by the genetic polymorphism of mbl gene to a great extent.Known mbl gene has a SNP G+230A who is positioned at the coding region, when genotype be+230G/A or+during 230A/A, serum MBL content can be wild gene type+230G/G serum content 10% or lower.The mbl gene promoter region has 2 SNP (G-550C and G-221C) can influence MBL content in the serum with G+230A in addition.For the haplotype of forming by G-550C (H/L), G-221C (Y/X) and these 3 SNP of G+230A (A/B), the MBL expression level is that high haplotype is to comprising HYA/HYA, HYA/LYA, HYA/LXA, LYA/LYA and LYA/LXA in the decision serum, the haplotype of MBL expression level in being comprises LXA/LXA, HYA/LYB and LYA/LYB in the decision serum, and the MBL expression level is that low haplotype comprises LXA/LYB and LYB/LYB in the decision serum.
Based on the biological function of above MBL, therefore we infer that MBL might be the tumor susceptibility gene that SARS-CoV infects.Polymorphism in the mbl gene influences MBL in the expression of serum level, and then causes the difference of the dependent SARS-CoV susceptibility infection of genotype.
The objective of the invention is by case-control study, analyze the above-mentioned pleomorphism site that determines its serum content in the mbl gene, to check between these pleomorphism sites and SARS-CoV susceptibility infection whether have dependency.
Summary of the invention
The contriver has analyzed the polymorphism that can determine the mbl gene of MBL expression level in the serum by case-control study, has investigated the dependency of these pleomorphism sites and the SARS-CoV susceptibility infection of mbl gene.By extensive case control study based on hospital, carried out a large amount of clinical and laboratory experiments and the statistical study of large sample, distinguish that the 230G/A that carries MBL or the genotypic individuality of 230A/A infect SARS-CoV higher susceptibility is arranged.Also distinguish simultaneously, in carrying or the individuality of the haplotype of low MBL expression level SARS-CoV is infected higher susceptibility is arranged (wherein, the haplotype of MBL expression level in being comprises LXA/LXA, HYA/LYB and LYA/LYB in the decision serum, and the MBL expression level is that low haplotype comprises LXA/LYB and LYB/LYB in the decision serum; These haplotypes are to be made up by the G-550C of mbl gene (H/L), G-221C (Y/X) and these 3 single nucleotide polymorphism (SNP) of G+230A (A/B) to form).
Therefore the present invention has identified a kind of new tumor susceptibility gene and tumor susceptibility gene type and the susceptible haplotype that SARS-CoV infects.
The present invention also provides a kind of susceptibility loci G+230A genotypic method related with the SARS-CoV susceptibility infection that detect, and this method is a PCR product sequencing.
The present invention also provide a kind of judge by the G+230A site of detecting mbl gene individual whether SARS-CoV is infected the method for susceptible.
The present invention also provides a kind of method that detects susceptible haplotype LXA/LXA, HYA/LYB, LYA/LYB, LXA/LYB and the LYB/LYB related with the SARS-CoV susceptibility infection, this method is calculated individual haplotype with the PHASE program then for determine the genotype of G-550C (H/L), G-221C (Y/X) and these 3 SNP of G+230A (A/B) with PCR product sequencing.
The present invention also provides a kind of haplotype LXA/LXA, HYA/LYB, LYA/LYB, LXA/LYB and LYB/LYB that forms by G-550 (H/L), G-221C (Y/X) and these 3 SNP of G+230A (A/B) of detecting by mbl gene to judge that whether individuality infects the method for susceptible to SARS-CoV.
The present invention also provides the primer that is used to detect mbl gene pleomorphism site G-550C, G-221C and G+230A right.
The present invention further provides and a kind ofly detected the test kit that infects other included 2 sites (G-550C and G-221C) of the G+230A site of related mbl gene and susceptible haplotype with SARS-CoV, and the mentioned reagent box prevent in the crowd and control the application of SARS-CoV infection.
One, as mentioned above, the invention provides a kind of method that detects the G+230A loci gene type of the mbl gene related with the SARS-CoV susceptibility infection, this method is a PCR product sequencing.Specifically, comprised following steps:
1), and sample genomic dna is carried out the specific PCR amplification of warm start and gradient to region, mbl gene G+230A site design gene order Auele Specific Primer;
2) the PCR product is checked order, distinguish the genotype in mbl gene G+230A site with this.
Two, the invention provides the 2 kind tumor susceptibility gene types related with the SARS-CoV susceptibility infection, promptly the 230G/A of mbl gene or 230A/A genotype are the tumor susceptibility gene type that SARS-CoV infects.And provide a kind of genotype to come judgement crowd or individual method to the SARS-CoV susceptibility infection by detection mbl gene G+230A site.This method comprises:
1), and sample genomic dna is carried out the specific PCR amplification of warm start and gradient to region, mbl gene G+230A site design gene order Auele Specific Primer;
2) the PCR product is checked order, distinguish the genotype in mbl gene G+230A site with this;
3) when the G+230A site of the mbl gene of a certain individuality shows as 230G/A or 230A/A genotype, show that then this individuality infects at SARS-CoV that to infect this viral risk when popular higher.
Three, the invention provides the 5 kind susceptible haplotypes related with the SARS-CoV susceptibility infection, promptly 5 kinds of haplotype combination such as mbl gene LXA/LXA, HYA/LYB, LYA/LYB, LXA/LYB or LYB/LYB are the tumor susceptibility gene type that SARS-CoV infects.And provide a kind of haplotype of forming by G-550C (H/L), G-221C (Y/X) and these 3 SNP of G+230A (A/B) of detecting by mbl gene to come judgement crowd or individuality method to the SARS-CoV susceptibility infection.Specifically, comprised following steps:
1), and sample genomic dna is carried out the specific PCR amplification of warm start and gradient to mbl gene G-550C (H/L), G-221C (Y/X) and this region, 3 sites design gene order Auele Specific Primer of G+230A (A/B);
2) the PCR product is checked order, distinguish the genotype in mbl gene G-550C (H/L), G-221C (Y/X) and these 3 sites of G+230A (A/B) with this;
3) calculate in each individuality G-550C (H/L), G-221C (Y/X) and these 3 haplotypes that the site is formed of G+230A (A/B) with the PHASE program by mbl gene.
Four, the invention provides a kind of method that detects haplotype LXA/LXA, HYA/LYB, LYA/LYB, LXA/LYB and the LYB/LYB of the mbl gene related with the SARS-CoV susceptibility infection, this method is to determine the genotype of G-550C (H/L), G-221C (Y/X) and these 3 SNP of G+230A (A/B) of mbl gene with PCR product sequencing, and calculates individual haplotype with the PHASE program.Specifically, comprised following steps:
1), and sample genomic dna is carried out the specific PCR amplification of warm start and gradient to mbl gene G-550C (H/L), G-221C (Y/X) and this region, 3 sites design gene order Auele Specific Primer of G+230A (A/B);
2) the PCR product is checked order, distinguish the genotype in mbl gene G-550C (H/L), G-221C (Y/X) and these 3 sites of G+230A (A/B) with this;
3) calculate in each individuality G-550C (H/L), G-221C (Y/X) and these 3 haplotypes that the site is formed of G+230A (A/B) with the PHASE program by mbl gene.
4) when some bodies show as LXA/LXA, HYA/LYB, LYA/LYB, LXA/LYB or LYB/LYB haplotype, show that then this individuality infects at SARS-CoV that to infect this viral risk when popular higher.
Five, the right sequence of Auele Specific Primer of detection mbl gene G+230A loci gene type is:
Forward primer: 5 '-CGGTCCCATTTGTTCTCACT-3 '
Reverse primer: 5 '-TCACAAACTGCTGTGTGGAAT-3 '
Detecting the G-550C of mbl gene and the right sequence of Auele Specific Primer of G-221C loci gene type is:
Forward primer: 5 '-AATGGGAGGAGGATTCAAGG-3 '
Reverse primer: 5 '-CCTFGTGACACTGCGTGACT-3 '
Six, the present invention further provides the test kit in other included 2 sites (G-550C and G-221C) of a kind of G+230A site of detecting the mbl gene related and susceptible haplotype with the SARS-CoV infection, one or more containers are housed in it, are equipped with in the container in order to detect one or more components of G-550C (H/L), G-221C (Y/X) and these 3 loci gene types of G+230A (A/B).Provide simultaneously with it can be through the audit of government's food and drug regulatory department, relevant medicine or biological products manufacturing, use and the information of sale aspect.For example, in the pcr amplification method of DNA is implemented, directly G-550C (H/L), the G-221C (Y/X) of test sample and the test kit of these 3 loci gene types of G+230A (A/B), the primer that contains PCR to (5 '-CGGTCCCATTTGTTCTCACT-3 ' and 5 '-TCACAAACTGCTGTGTGGAAT-3 '; 5 '-AATGGGAGGAGGATTCAAGG-3 ' and 5 '-CCTTGTGACACTGCGTGACT-3 '), dNTPs is used for the archaeal dna polymerase of PCR reaction and damping fluid thereof etc.It is known to those skilled in the art that above component only is schematically, the archaeal dna polymerase of the described PCR of being used for reaction can be the TaqDNA polysaccharase, for example, HotStar Taq enzyme, Klenow fragment, the Tth archaeal dna polymerase, VENTDNA polysaccharase etc. can be used in the enzyme of pcr amplification.But for guaranteeing specific amplification, except the optimization of primer and reaction conditions, preferred HotStarTaq enzyme.
Using method is described as follows:
To sample gene group DNA (its concrete preparation method is referring to embodiment), use 2 pairs of primers provided by the invention respectively, carry out pcr amplification according to following reaction system and condition: 12.5-μ l reaction system is: 20～100ng genomic dna, 0.2 μ M primer, 0.2mM dNTP, 1.5mM MgCl
2, 1.0unit HotStarTaq
TMArchaeal dna polymerase and 1 * damping fluid (QIAGEN, Chatsworth, CA).Carry out the amplification in 2 stages after 95 ℃ of pre-sex change of 15min, its sex change and extension term harmonization, be 94 ℃ of 30s and 72 ℃ of 50s, in 13 circulations of fs, annealing temperature is since 0.5 ℃ of 62 ℃ of each cycle down, the annealing temperature of subordinate phase amplification then is fixed on 56 ℃, and 72 ℃ of last extension times are 7min.The PCR product checks order through routine, and figure judges genotype according to the peak.
Seven, the invention provides the detection method and the application in prevention of SARS-CoV infection population and control thereof in other included 2 sites (G-550C and G-221C) of mbl gene G+230A site related and susceptible haplotype with the SARS-CoV susceptibility infection.For example, because the present invention has confirmed mbl gene G+230A site, or by G-550C (H/L), the haplotype combination LXA/LXA that G-221C (Y/X) and G+230A (A/B) form in these 3 sites, HYA/LYB, LYA/LYB, LXA/LYB or LYB/LYB, exist related with the SARS-CoV susceptibility infection, therefore, in the prevention and control of SARS-CoV infection population, just need carry out the G-550C (H/L) of mbl gene to the object of being seeked advice from, the genotype of G-221C (Y/X) and G+230A (A/B) is analyzed, to judge whether this object carries SARS-CoV and infect tumor susceptibility gene type 230G/A or 230A/A, perhaps carries susceptible haplotype combination LXA/LXA, HYA/LYB, LYA/LYB, LXA/LYB or LYB/LYB.To having mbl gene 230G/A or 230A/A genotype, the individuality (show as infect to have higher susceptibility to SARS-CoV) that perhaps has susceptible haplotype combination LXA/LXA, HYA/LYB, LYA/LYB, LXA/LYB or LYB/LYB, then can carry out the intervention of science to it, comprise that the therapeutic prevention is carried out in suggestion early or, can reduce the risk of the SARS-CoV infection of these Susceptible population so pointedly if might then inoculate SARS vaccine etc.
Embodiment:
Embodiment 1
This embodiment has designed and synthesized 2 pairs of primers, and on the right basis of this primer, designed a kind of G+230A site of detecting the mbl gene related with the SARS-CoV susceptibility infection, and the method for the susceptible haplotype combination LXA/LXA, HYA/LYB, LYA/LYB, LXA/LYB or the LYB/LYB that form by G-550C (H/L), G-221C (Y/X) and these 3 SNP sites of G+230A (A/B).
1, the extraction of genomic dna
Take the Miller improvement salting-out process of this area routine to extract the peripheral blood leucocyte genomic dna.
1) gets Trisodium Citrate or EDTA-Na
2Anticoagulated whole blood 5ml (as far as possible without anticoagulant heparin) places 50ml to cover centrifuge tube;
2) add 3-5 times of volume cold distilled water, put upside down mixing repeatedly, placed 5 minutes in the ice, centrifugal 20 minutes of 4 ℃ of 2000rpm;
3) slowly topple over supernatant, precipitation adds the 0.1%Triton-X100 (volume is the same) of precooling, and soft mixing precipitation is as above centrifugal, abandons supernatant;
4) (50Mm Tris-Cl-10mM EDTA pH8.0), thoroughly smashes precipitation to precipitation adding 4ml lysate, and adding 10%SDS then is 0.5% to final concentration, mixing;
5) adding Proteinase K (10mg/ml) is 200 μ g/ml to final concentration, mixing, 55 ℃ of 3 hours or 37 ℃ of digested overnight (is good with digested overnight);
6) add 6M NaCl 1.2ml, thermal agitation 20 seconds, 2, centrifugal 10 minutes of 200rpm is transferred to another with supernatant and covers centrifuge tube;
7) add 2 times of volume precooling dehydrated alcohols, put upside down mixing repeatedly, choose DNA to another Eppendorf pipe to cotton-shaped DNA precipitation occurring;
8) precipitate 2 times with 75% pre-cooled ethanol washing DNA;
9) with 0.5ml TE (10mM Tris-Cl-EDTA, pH8.0) or dissolved in distilled water DNA precipitation, electrophoresis detection dna fragmentation size, or survey 260/280nm OD ratio.
2, pcr amplification
Use 2 pairs of primers provided by the invention respectively, carry out pcr amplification according to following reaction system and condition: 12.5-μ l reaction system is: 20～100ng genomic dna, 0.2 μ M primer, 0.2mM dNTP, 1.5mM MgCl
2, 1.0unitHotStarTaq
TMArchaeal dna polymerase and 1 * damping fluid (QIAGEN, Chatsworth, CA).Carry out the amplification in 2 stages after 95 ℃ of pre-sex change of 15min, its sex change and extension term harmonization are 94 ℃ of 30s and 72 ℃ of 50s, in 13 circulations of fs, annealing temperature is since 0.5 ℃ of 62 ℃ of each cycle down, and the annealing temperature of subordinate phase amplification then is fixed on 56 ℃.72 ℃ of last extension times are 7min.The PCR product is identified the genotype in G-550C (H/L), G-221C (Y/X) and these 3 sites of G+230A (A/B) with sequencing.
3, haplotype calculates
For the genotypic individuality of measuring G-550C (H/L), G-221C (Y/X) and these 3 sites of G+230A (A/B), use the PHASE program and calculate its corresponding haplotype.
Embodiment 2
The method that this embodiment sets up with embodiment 1 is analyzed G-550C (H/L), the G-221C (Y/X) of the mbl gene of 352 routine SARS patients, 392 routine normal healthy controls individualities and the genotype in these 3 sites of G+230A (A/B).
1, subjects:
During year July in May, 2003 to 2003, in the ward of the Beijing Xiaotangshan Hospital, collect SARS patient's 352 examples that meet WHO standard.Among this 352 routine patient, 52 examples suffer from some basic diseases that may increase the SARS risk, (are P as important diseases such as diabetes, hypertension, heart trouble, pulmonary tuberculosis, asthma and tumours
IGroup); Other has 20 examples is to suffer from the patient of serious SARS, and they move in intensive care unit(ICU) (ICU) or death (is P
IIGroup); Remaining 280 examples are P
IIIGroup.Other collects age, sex and ethnic healthy individual 392 examples of mating crowd in contrast.Sample collection process is carried out according to the national human genome criterion that studies ethics, and has obtained to be raised the informed consent of object.
2, determine the genotype in mbl gene G-550C (H/L), G-221C (Y/X) and these 3 sites of G+230A (A/B) and the haplotypes that constitute of 3 sites thus:
The genotype frequency in mbl gene G-550C (H/L), G-221C (Y/X) and these 3 sites of G+230A (A/B) distributes as shown in table 1 in 352 routine SARS patients and the 392 routine normal healthy controls individualities.
Work as P
I+ P
II+ P
IIIWhen comparing, the frequency of 230G/G is respectively 65.6% and 76.8% (the OR value is 1.73) in SARS patient and normal healthy controls, the MBL expression level is that the frequency of high haplotype is respectively 64.0% and 74.5% (the OR value is 1.67) in SARS patient and normal healthy controls in the decision serum; Work as P
II+ P
IIIWhen comparing, the frequency of 230G/G in SARS patient and normal healthy controls is respectively 64.3% and 76.8% (the OR value is 1.84); The MBL expression level is that the frequency of high haplotype combination is respectively 63.1% and 74.5% (the OR value is 1.75) in SARS patient and normal healthy controls in the decision serum.Therefore, the G+230A pleomorphism site of MBL and the haplotype combination of forming by G-550C (H/L), G-221C (Y/X) and these 3 sites of G+230A (A/B) and the dependency of SARS susceptibility infection have been resolved with method of the present invention.
Should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, but the equivalent form of value doing to change or revise drop on equally within the application's letter of authorization institute restricted portion.
CI: fiducial limit.Whether related by carrying out logistic regression analysis pleomorphism site with the susceptibility of SARS infection, proofread and correct these two kinds of risk factors of age and sex during analysis, and carried out the multiple check correction.
aThe MBL expression level is that high haplotype combination comprises HYA/HYA, HYA/LYA, HYA/LXA, LYA/LYA and LYA/LXA in the decision serum, the haplotype combination of MBL expression level in being comprises LXA/LXA, HYA/LYB and LYA/LYB in the decision serum, and the MBL expression level is that low haplotype combination comprises LXA/LYB and LYB/LYB in the decision serum.
bG-550C:G/G genotype and G/C+C/C genotype are relatively; G-221C:G/G genotype and G/C+C/C genotype are relatively; G+230A:G/G genotype and G/A+A/A genotype are relatively; Haplotype: Gao Yuzhong+relatively low.
The number of somatotype sample changes to some extent because of the failure of the individual somatotype of minority.
Reference
1.Zhang?H，Zhou?G，Zhi?L，Yang?H，Zhai?Y，Dong?X，Zhang?X，He?F.Association?ofmannose-binding?lectin?gene?polymorphisms?with?susceptibility?to?severe?acute?respiratorysyndrome?coronavirus?infection.J?Infect?Dis?2005；In?press.
2.Rota?PA，Oberste?MS，Monroe?SS，etal.Characterization?of?a?novel?coronavirus?associated?withsevere?acute?respiratory?syndrome.Science?2003；300：1394-9.
3.Lee?N，Hui?D，Wu?A，et?al.A?major?outbreak?of?severe?acute?respiratory?syndrome?in?HongKong.N?Engl?J?Med?2003；348：1986-94.
4.Ho?KY，Singh?KS，Habib?AG，et?al.Mild?illness?associated?with?severe?acute?respiratorysyndrome?coronavirus?infection：lessons?from?a?prospective?seroepidemiologic?study?ofhealth-care?workers?in?a?teaching?hospital?in?Singapore.J?Infect?Dis?2004；189：642-7.
5.Booth?CM，Matukas?LM，Tomlinson?GA，et?al.Clinical?features?and?short-term?outcomes?of144?patients?with?SARS?in?the?greater?Toronto?area.JAMA?2003；289：2801-9.
6.Ng?MH，Lau?KM，Li?L，et?al.Association?of?human-leukocyte?-antigen?class?I(B*0703)andclass?II(DRB1*0301)genotypes?with?susceptibility?and?resistance?to?the?development?of?severeacute?respiratory?syndrome.J?Infect?Dis?2004；190：515-8.
7.Holmskov?U，Thiel?S，Jensenius?JC.Collections?and?ficolins：humoral?lectins?of?the?innateimmune?defense.Annu?Rev?Immunol?2003；21：547-78.
8.Turner?MW.The?role?of?mannose-binding?lectin?in?health?and?disease.Mol?Immunol2003；40：423-9.
9.Bisht?H，Roberts?A，Vogel?L，et?al.Severe?acute?respiratory?syndrome?coronavirus?spike?proteincxpressed?by?attenuated?vaccinia?virus?protectively?immunizes?mice.Proc?Natl?Acad?Sci?USA2004；101：6641-6.
Sequence table
＜110〉INST OF EMISSION ﹠ RADIATION M
＜120〉a kind of method and MBL tumor susceptibility gene that infects relevant tumor susceptibility gene mbl gene type with SARS-CoV that detect
<160>4
<210>1
<211>20
<212>DNA
＜213〉artificial sequence
<400>1
CGGTCCCATT?TGTTCTCACT 20
<210>2
<211>21
<212>DNA
＜213〉artificial sequence
<400>2
TCACAAACTG?CTGTGTGGAA?T 21
<210>3
<211>20
<212>DNA
＜213〉artificial sequence
<400>3
AATGGGAGGA?GGATTCAAGG 20
<210>4
<211>20
<212>DNA
＜213〉artificial sequence
<400>4
CCTTGTGACA?CTGCGTGACT 20
Claims (5)
1. a detection and SARS-CoV infect the included G-550C of the G+230A site of related mbl gene and susceptible haplotype and the test kit in G-221C site, comprise: amplification shown in the sequence 1～4 with gene-specific primer, dNTP, be used for one or more of archaeal dna polymerase that PCR reacts and damping fluid thereof, and one or more of the reagent of the usefulness that is used for checking order.
2. detection kit according to claim 1, it is characterized by described archaeal dna polymerase is HotStar Taq enzyme.
3. an Auele Specific Primer that detects mbl gene G+230A loci gene type is right, it is characterized by:
Forward primer: 5 '-CGGTCCCATTTGTTCTCACT-3 '
Reverse primer: 5 '-TCACAAACTGCTGTGTGGAAT-3 '.
4. the Auele Specific Primer of G-550C who detects mbl gene and G-221C loci gene type is right, it is characterized by:
Forward primer: 5 '-AATGGGAGGAGGATTCAAGG-3 '
Reverse primer: 5 '-CCTTGTGACACTGCGTGACT-3 '.
5. claim 3 or 4 described primers is characterized by this detection reagent and can detect the G+230A site or the included G-550C and the G-221C site of susceptible haplotype of infecting related mbl gene with SARS-CoV the application in the preparation detection reagent.
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| CN111455062B (en) * | 2020-04-01 | 2022-02-11 | 中国人民解放军总医院 | Kit and platform for detecting susceptibility genes of novel coronavirus |
| EP4288079A1 (en) * | 2021-02-08 | 2023-12-13 | Humanitas Mirasole S.p.A. | Molecules for use in the treatment and/or prevention of covid-19 |
Citations (3)
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| CN1468965A (en) * | 2003-05-16 | 2004-01-21 | 浙江大学医学院附属第一医院 | Gene detection reagent kit for SARS virus and its detection method |
| CN1528915A (en) * | 2003-04-26 | 2004-09-15 | 杭州华大基因研发中心 | Method for quickly detecting single base polymorphism of SARS virus |
| WO2004089394A2 (en) * | 2003-04-09 | 2004-10-21 | Natimmune A/S | Treatment of severe acute respiratory syndrome (sars) |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004089394A2 (en) * | 2003-04-09 | 2004-10-21 | Natimmune A/S | Treatment of severe acute respiratory syndrome (sars) |
| CN1528915A (en) * | 2003-04-26 | 2004-09-15 | 杭州华大基因研发中心 | Method for quickly detecting single base polymorphism of SARS virus |
| CN1468965A (en) * | 2003-05-16 | 2004-01-21 | 浙江大学医学院附属第一医院 | Gene detection reagent kit for SARS virus and its detection method |
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| W. K. Eddie lp et al..Mannose-Binding Lectin in Severe Acute RespiratorySyndrome Coronavirus Infection.The Journal of Infectious Diseases191.2005,1911697-704. * |
| Yu Lung Lau, JS Malik Peiris.Pathogenesis of severe acute respiratory syndrome.Current Opinion in Immunology17.2005,17404-410. * |
| 周钢桥,贺福初.人类传染性疾病的遗传易感性.基础医学与临床24 5.2004,24(5),491-496. * |
| 高洁,李静,娄晋宁.SARS冠状病毒的研究进展.中日友好医院学报19 3.2005,19(3),178-183. |
| 高洁,李静,娄晋宁.SARS冠状病毒的研究进展.中日友好医院学报19 3.2005,19(3),178-183. * |
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