CN1477209A - Method for quickly-detecting hepatilis C virus and its gene type - Google Patents

Abstract

The present invention relates to a method for quickly detecting hepatitis C virus and its gene type. The oligonucleotide probe is fixed on the treated slide, after the specific combination of it and digoxin or biotin-labeled PCR product the alkaline phosphatase labeled anti-digoxin antibody or avidin-labeled enzyme-linked antibody can be added to make it combine with hybridization product with digoxin or biotin, on the correspondent type specific probe position a blue point is formed, so that the HCV positive reaction and its gene type can be quickly defined.

Description

A kind of rapid detection hepatitis C virus and genotypic method thereof

Technical field

The present invention relates to a kind of easy, the cheap hepatitis C virus diagnosis and the making method and the application method thereof of somatotype detection chip, is a kind of method and application thereof of hepatitis C virus and genotype thereof being carried out rapid detection from the gene angle specifically.

Background technology

(1) hepatitis C (third liver) is a kind of disease of wide-scale distribution, it is the main diseases therefore that causes chronic viral hepatitis, liver cirrhosis, hepatocellular carcinoma (HCC), and these pathologies form relevant (Amarapurkar D.Natural history of hepatitis C virus infection.JGastroenterol Hepatol with its some important biological characteristics of cause of disease hepatitis C virus (HCV), 2000,159 (suppl): E105-110).Ren Tong HCV strain mainly is divided into six genotype and more than 100 gene hypotype (Foms, X., and J.Bukh.Mcthods for dctermining hepatitis Cvirus genotype.Viral Hep.Rev, 1998,4:1-19 in the world; Foms, X., M.D Maluenda, F.X.Lopez-Labrador, S.Ampurdanes, E.Olmedo, J.Costa, P.Simmonds.Comparative study of three methods for genotyping hepatitis C virus strains insamples from Spanish patients.J.Clin.Microbiol.1996.34:2516-2521).And the distribution of type and region have much relations (Simmonds P.Variability of hepatitis C virus.Hepatology, 1995,21:570-583).1a type preponderate in the U.S., Brazil and Northern Europe (GaryL.Davis.The American Journal of Medicine.December 27,1999 Volume 107 (6B)) wherein.The 1b type in the Far East, European the western and eastern sees more, 2a and 2b type are distributed in Asia and Europe, the 3a type is more in west and Thailand, Singapore, and the Middle East, the common gene hypotype in South Africa and area, Hong Kong are respectively 4a, 5a and 6a, and China is mainly based on 1b and 2a type (Jiang Weilun, Gu officers and people etc.1997 the 9th the 4th phases of volume of Shanghai preventive medicine magazine).

Some clinical studies data show that the HCV genotype not only affects the clinical course of hepatitis C, and also closely related with antiviral therapy, and Interferon, rabbit (IFN) is the present modal antiviral biotechnological formulation that is used for treating hepatitis c.The reply situation of the HCV of different genotype to the IFN treatment reported in some researchs.People such as Chemello studies show that (Chemello L.Hepatitis C serotypeand response to interferon therapy.N Engl J Med, 1994,330:143), HCV was genotypic before the long-term responsiveness of IFN treatment and unresponsiveness were treated to a great extent influences.The long-term response rate of HCVI, II, III type is respectively 29%, 52%, 74%, and visible III type HCV is higher to the IFN response rate.Most scholars think that the HCV genotype is the good predict index of IFN result of treatment, have important directive significance for the treatment of chronic hepatitis C.

The diagnosis of hepatitis C and treatment mainly depend on serum whose anti-HCV detection of antibodies, along with deepening continuously that HCV is familiar with, it is found that serology detection sensitivity specificity is relatively poor, can't determine the virogene type and the prediction state of an illness, judge curative effect.

Serotype (Pawlotsky J.M, Prescott L, Simmond P, et al.Comparison with astandardized genotyping assay[J] .J Clin Microbiol, 1997,35:1734-39.)): be according to HCV C district or NS4 district different shaped or hypotype synthetic peptide as the antigen of somatotype, detect the Idiotype antibody in patient's serum.Advantages such as it is easy, quick, economic, accurate that serotype has, shortcoming is: result's accuracy is than gene type difference, cross reaction takes place between the different shaped can cause the somatotype mistake, in some cases, the specific antibody that may not contain at designed polypeptide in the tested serum causes false negative, and serological typing insufficient sensitivity in some immune deficiencies or in the infected of immunosuppressor, may cause not having the generation of HCV specific antibody owing to lack immunne response in the body.

The method that adopts for conventional H CV gene type mainly contains: restriction fragment length polymorphism analysis (RFLP) somatotype, type specificity PCR primer somatotype, direct order-checking etc.

1, restriction fragment length polymorphism analysis (RFLP) somatotype (Davidson F.Simmonds P, Ferguson J C, et al.Surverof major genotypes and subtypes of hepatitis C virususing RFLP of sequences amplified from the 5 non-coding region[J] .J GenVirol, 1995,76:1197-1204 :) utilize restriction enzyme to discern specific restriction enzyme site in RT-PCR amplification HCV5 '-UTR district product, carry out enzyme and cut, cut the size of product according to enzyme and judge the HCV genotype.The PFLP typing has correctly, quick, economic dispatch advantage, but needs to improve so that can distinguish more variation.

2, type specificity PCR primer somatotype (Okamato H.Kojima M.Sakamoto M, et al.Theentire nucleotide sequence and classification of a hepatitis C virus isolate of anovel genotype from an Indonesian patient with chronic levre disease[J] .J GenVirol, 1994,75:629-635.)) according to the difference of different HCV types, design a series of Auele Specific Primers at a certain sector sequence.Different shaped can amplify different fragments, and carries out somatotype with this because gene order variation is bigger between the hypotype of coding region, each type and hypotype thereof can be fine separately, but various design of primers is had relatively high expectations, guarantee has than big-difference between various.

3, directly order-checking is more accurate than other two kinds of methods, but this method is time-consuming, expensive, is not suitable for a large amount of somatotype work, and the somatotype of sensitivity carry out to(for) polyinfection person is lower, unless adopt clone and the method for measuring cDNA to identify preponderant genotype HCV.

At present domestic for the hepatitis C gene type, still there is not the standard reagent box of generally applying.Using more is Belgian INNOGENETICS company improved recently s-generation spectral line probe analysis test kit, but cost is relatively more expensive, and operates loaded down with trivial details.

Utilize gene chip to carry out that HCV detects and somatotype has overcome the defective of above-mentioned certain methods, and can carry out accurate somatotype and have characteristics such as highly sensitive, high specificity polyinfection person.Gene chip hybridization result's detection method mainly is fluorescent marker method and isotope-labelling method, fluorescent marker method is little with himself peculiar toxic side effect, highly sensitive, can various product parallel processing (different fluorescence molecules has different wavelength, available laser confocal scanning instrument scans simultaneously the fluorescence of different wave length and reaches the purpose that various product detect simultaneously) etc. advantage take the course of its own, obtain people from all walks of life's favor.But because its signal fetch equipment costs an arm and a leg, make this technology be difficult to apply socially, developing country especially, thus restricted the application of chip technology aspect medical clinic applications greatly.China also only has minority scientific research tissue and company to possess the ability of this equipment of outfit at present.

We utilize development process to carry out signal detection, can use cheap ordinary optical scanner to scan on the one hand, and gained collection of illustrative plates input computer is carried out image analysis and data preparation work; Under the less situation of quantity of information, also can use magnifying glass or bore hole direct viewing on the other hand, thereby provide favourable condition for gene chip diagnosis technology applying in the whole society.Thereby be guided out purpose of the present invention.

Summary of the invention

The object of the invention provides rapid detection hepatitis C virus (HCV) and genotypic method thereof, specifically, oligonucleotide probe is fixed on the treated slide, the PCR product of digoxin (or vitamin H) mark with it specificity in conjunction with after, the anti digoxin antibody (or enzyme labelled antibody of avidin mark) that adds alkali phosphatase enzyme mark then, the latter combines with the hybridization product of band digoxin (or vitamin H), blue dot occurs on corresponding type specificity probe location.Determine genotype and the hypotype of HCV according to a position.From PRELIMINARY RESULTS, this law is highly sensitive, high specificity, simple to operate, in 7-8 hour, can finish PCR, hybridization, testing, can differentiate the difference of single base, in addition with the naked eye, magnifying glass or microscope be with regard to the decidable result, do not need expensive experimental equipment.Can be used for detecting HCV RNA and its hypotype, have clinic popularization and application values.

Another object of the present invention provides making method, the sample treatment of said chip.

Making method involved in the present invention is as follows:

(1) design of probe and preparation

(2) processing of sample and digoxigenin labeled pcr amplification

(3) hybridization, detection and interpretation of result 1, probe design

According to hepatitis C virus gene 5 '-UTR is the characteristics of the sequence information at conservative position in the HCV genome, design corresponding probe, principle is the centre that each mutational site is positioned at probe as far as possible, the length of each probe is identical, modify on the good slide in order to be fixed on better, the end of 5 of probe ' need add the connecting arm of 16 length T and amido modified.

Table 1: according to the designed probe of somatotype information of the dna sequence dna of hepatitis c virus gene 5 '-UTR

Numbering (Number)	Genotype and hypotype (Type)	Probe sequence (sequence from 5 ' to 3 ')
			1	General	TTG?GGC?GTG?CCC?CCG?C
2	TCT?GCG?GAA?CCG?GTG?A
		3	The I type		AAT?TGC?CAG?GA(C/T)GAC C
4	TCT?CCA?GGC?ATT?GAG?C

????5	??1a/2a	CCC?CGC?AAG?ACT?GCT?A
			????6	??1a/1b	CCG?CAA?GAC?CGC?TAG?C
????7	??1b	CCG?CGA?GAC?CGC?TAG?C
			????8	??1b	CCG?CGA?GAC?TGC?TAG?C
????9	The II type	TAG?CGT?TGG?GTT?GCG?A
			????10	ATA?GAG?TGG?GTT?TAT?C
????11		??2a			CCG?GGA?AGA?CTG?GGT?C
	????12		ACC?CAC?TCT?ATG?CCC?G
????13				??2b	CCG?GAA?AGA?CTG?GGT?C
	????14	ACC?CAC?TCT?ATG?TCC?G
????15			The III type		AAT?CGC?TGG?GGT?GAC?C
	????16	TIT?CTG?GGT?ATT?GAG?C
????17				??IV/3b	TTT?CCG?GGC?ATT?GAG?C
	????18	The IV type	AAT?CGC?CGG?GAT?GAC?C
????19				??3a	CCG?CGA?GAT?CAC?TAG?C
	????20	??6a	GGG?TCC?TTT?CCA?TTG?G

2, the preparation of chip

After probe synthesizes well, with deionized water with probe dilution, and mix with spoting solution equal-volume, making final concentration is 75pmols/ μ l,, place under 70% humidity, the room temperature condition and to fix in 48-72 hour in aldehyde group modified slide surface by Cartesian microarray manufacturing system dot matrix.3, sample preparation and mark

(1) extracting of serum RNA

5ul sample serum is added on the 0.5ml Eppendorf pipe end, adds 10ul GeneReleaserTM again, avoids the mixing that vibrates, shop one deck paraffin oil is put into the pcr amplification instrument, carries out the gene release procedure: 65 ℃ * 30 seconds, 8 ℃ * 30 seconds, 65 ℃ * 90 seconds, 97 ℃ * 3 minutes, 8 ℃ * 60,65 * 3 minutes, 97 ℃ * 60 seconds, 65 ℃ * 60 seconds, 80 ℃ * 10 minutes.(2) reverse transcription and first round PCR (RT-PCR single stage method)

70 ℃ of heating of 5ul reverse transcriptase primer liquid (containing reverse transcriptase primer 10pmol, RNasin 20U) 5 minutes, ice bath adds 10ul reverse transcription amplification liquid and [contains overcoat forward primer (P immediately ₃, P ₄) 10pmol, dNTP 0.2mM, 1 * PCR Buffer, MgCL22Mm, AMV 4U, Taq enzyme 5U).The amplification pipe places the pcr amplification instrument, and 42 ℃ of reverse transcriptions 50 minutes are then carried out amplification program: 94 ℃ * 30 seconds, and 55 ℃ * 30 seconds, 72 ℃ * 40 seconds, 30 circulations.(3) second take turns PCR

3ul first round PCR product and 27ul second take turns the PCR reaction solution and mix and [contain P ₁, P ₂Each 10pmol of primer (digoxigenin labeled), dNTP 0.2Mm, 1 * PCR Buffer, 2mM MgCI2, Taq E enzyme 1.5U] the same PCR for the first time of pcr amplification cycling program.

Table 2: HCV 5 '-UTR dna sequence dna is carried out the required primer of pcr amplification

Numbering numb er	Primer sequence (sequence from 5 ' to 3 ')	??Tm( ??℃)
			P1	?5′-CACTCGCAAGCACCCTATCAGGC ?AGT-3′	??71
P2	?5′-TCTAGCCATGGCGTTAGTA(C/T)G ?AGTGT-3′	??65
			P3	?5′-GCTCATG(G/A)TGCACGGTCTAC ?GAGACCT-3′	??70
P4	?5′-CCCTGTGAGGAACT(A/T)CTGTC ?TTCACGC-3′	??67

(4) chip hybridization and result detect

Get the PCR product 2 μ l of mark, 98 ℃ of sex change 4mins, then with 20 μ l Easy-Hb hybridization solution mixings, getting 10 μ l drips in the chip surface that has fixed, covered places 42 ℃ of wet boxes to hybridize 25mins, puts then to swing among the washing lotion I and washes 10mins, airing is at last with GenePi 4000B scanning of GenePix Pro chip signal analytical system and analytical results.This shows that advantage of the present invention and effect are conspicuous:

1, whether detection method of the present invention not only can qualitative detection have hepatitis C virus, and can detect the genotyping information of hepatitis C virus.

2, detection method of the present invention has the high characteristics of detection sensitivity, can differentiate the difference of single base;

3, the observation of detection of the present invention and analytical procedure is characterized in that: can be by magnifying glass and microscopic examination and plain scan instrument scanning analysis result etc., and with low cost, make the more convenient easy row of clinical application

Description of drawingsFig. 1 comprises the hepatitis C virus detection of 20 probes and the result that Genotyping Chip is used to detect actual sample.

EmbodimentEmbodiment 1, probe design and chip manufacturing

According to hepatitis C virus gene 5 '-UTR is the situation of the sequence information at conservative position in the HCV genome, the probe of design length identical (16mer).As shown in table 3 below:

The probe table 3 that table 3. is designed according to the somatotype information of the dna sequence dna of hepatitis c virus gene 5 '-UTR

Numbering (Number)	Genotype and hypotype (Type)	Probe sequence (sequence from 5 ' to 3 ')
			????1	General	TTG?GGC?GTG?CCC?CCG?C
????2	TCT?GCG?GAA?CCG?GTG?A
		????3	The I type		AAT?TGC?CAG?GA(C/T)GAC C
????4	TCT?CCA?GGC?ATT?GAG?C
		????5		??1a/2a	CCC?CGC?AAG?ACT?GCT?A
????6	??1a/1b		CCG?CAA?GAC?CGC?TAG?C
		????7		??1b	CCG?CGA?GAC?CGC?TAG?C
????8	??1b		CCG?CGA?GAC?TGC?TAG?C
		????9		The II type	TAG?CGT?TGG?GTT?GCG?A
????10	ATA?GAG?TGG?GTT?TAT?C
		????11	??2a		CCG?GGA?AGA?CTG?GGT?C
????12	ACC?CAC?TCT?ATG?CCC?G
		????13		??2b	CCG?GAA?AGA?CTG?GGT?C
????14	ACC?CAC?TCT?ATG?TCC?G
		????15	The III type		AAT?CGC?TGG?GGT?GAC?C
????16	TTT?CTG?GGT?ATT?GAG?C
		????17		??IV/3b	TTT?CCG?GGC?ATT?GAG?C
????18	The IV type		AAT?CGC?CGG?GAT?GAC?C
		????19		??3a	?CCG?CGA?GAT?CAC?TAG?C
????20	??6a		?GGG?TCC?TTT?CCA?TTG?G

The synthetic oligonucleotide is soluble in water, mix with the Spoting Solution of equal volume then, making final concentration is 75pmol/ μ l, uses Cartesian chip manufacturing system dot matrix in aldehyde group modified slide surface, place under the room temperature, 70% humidity is preserved and was fixed in 48-72 hour.The extracting of the processing of embodiment 2:HCV sample and mark (1) serum RNA

5ul sample serum is added on the 0.5ml Eppendorf pipe end, adds 10ul GeneReleaserTM again, avoids the mixing that vibrates, shop one deck paraffin oil is put into the pcr amplification instrument, carries out the gene release procedure: 65 ℃ * 30 seconds, 8 ℃ * 30 seconds, 65 ℃ * 90 seconds, 97 ℃ * 3 minutes, 8 ℃ * 60,65 * 3 minutes, 97 ℃ * 60 seconds, 65 ℃ * 60 seconds, 80 ℃ * 10 minutes.(2) reverse transcription and first round PCR (RT-PCR single stage method)

70 ℃ of heating of 5ul reverse transcriptase primer liquid (containing reverse transcriptase primer 10pmol, RNasin 20U) 5 minutes, ice bath adds 10ul reverse transcription amplification liquid and [contains overcoat forward primer (P immediately ₃, P ₄) 10pmol, dNTP 0.2mM, 1 * PCR Buffer, MgCL22Mm, AMV 4U, Taq enzyme 5U].The amplification pipe places the pcr amplification instrument, and 42 ℃ of reverse transcriptions 50 minutes are then carried out amplification program: 94 ℃ * 30 seconds, and 55 ℃ * 30 seconds, 72 ℃ * 40 seconds, 30 circulations.(3) second take turns PCR

3ul first round PCR product and 27ul second take turns the PCR reaction solution and mix and [contain P ₁, P ₂Each 10pmol of primer (digoxigenin labeled), dNTP 0.2Mm, 1 * PCR Buffer, 2mM MgCI2, Taq E enzyme 1.5U] the same PCR for the first time of pcr amplification cycling program.The PCR product of embodiment 3, digoxigenin labeled and the hybridization of chip

After 98 ℃ of sex change in 4 minutes of PCR product 2 μ l with digoxigenin labeled, placed immediately 4 minutes on ice.Mix with 20 μ l tetramethyl phosphonium chloride amine hybridization solutions then, drip in chip surface.Covered is in 42 ℃ of hybridization 30min, at room temperature, take out chip, remove cover glass, drop among the washing lotion I (containing chlorination tetramethyl-amine) shaking table rapidly and swing and wash 10mins, then chip is put into washing lotion II balance 1min, take out chip again, absorb unnecessary liquid with thieving paper, other gets 20 μ l antibody-solutions and drips in chip surface, and covered leaves standstill 30mins, antibody is fully combined, the albumen nonspecific binding site on the chip of blockading simultaneously with digoxin or vitamin H.

Remove cover glass, absorb unnecessary liquid, put then to swing among the washing lotion II and wash 1min, take out chip, absorb unnecessary liquid with thieving paper.

Embodiment 4: film transfer printing colour developing

Get 30 μ l balance drops in chip surface, left standstill 1 minute, absorb unnecessary liquid, the nylon membrane of getting a 1 * 1cm2 then soaks into colour developing liquid bonnet in chip surface, and black out left standstill 20-30 minute.

Take the nylon membrane of chip surface off, and dry or dry once with deionized water rinsing.Embodiment 5: signal detection and the analysis information on the ordinary optical scanning instrument record nylon membrane.(result as shown in Figure 1).

Obtain corresponding somatotype information according to results of hybridization, the sequencing result with respective sample compares (shown in the table 4) then.(the sample order-checking entrusts Bo Ya company to finish)

Table 4. compares the third hepatopathy human sample somatotype result and sequencing result with chip detecting method of the present invention

Sample is compiled chip somatotype sequencing result genotype

Number result

1.8????????3b/1b????????-138?????????????3b

GGA ACAACC??????1b

-100

CCGC GAGAC

1.10???????2a/2c?????????-167????????????2a/2c

TGCC GGGA AGA

-81

TAG CGTTGGG

3.3?????3b?????-167??????????????????3b

AAT CGCC GGGA??????3b

-100

CGC GAGA TCAC

3.4?????1b?????-100??????????????????1b

CCGC GAGA???????????1b

-220

AGGA TCCC

3.5?????1b?????-100??????????????????1b

CCGC GAGA

5.10????6a?????-147??????????????????6a

CCTTTC(CA)TTG

G

By table 4 as seen, the coincidence rate of chip detection result and sequencing result is 100%.This shows that detect hepatitis C virus (HCV) gene type susceptibility with the third liver gene somatotype detection chip and can reach more than 90%, specificity is 100%.

Claims (5)

1. a rapid detection hepatitis C virus and genotypic method thereof, it is characterized in that oligonucleotide probe is fixed on the treated slide, digoxin or biotin labeled PCR product with it specificity in conjunction with after, add the anti digoxin antibody of alkali phosphatase enzyme mark or the enzyme labelled antibody of avidin mark then, make it to combine with hybridization product with digoxin or vitamin H, on corresponding type specificity probe location, blue dot occurs, thereby determine the HCV positive and genotype fast.

2. by described rapid detection hepatitis C virus of letter of authorization requirement and genotypic method thereof, it is characterized in that described surface of glass slide is through aldehyde group modified processing.

3. one kind is used for rapid detection hepatitis C virus and genotypic chip preparation method thereof, comprises probe design, chip preparation, sample preparation and mark, chip hybridization and detection, it is characterized in that:

(1) 5 ' of probe end need add the connecting arm of 16 length T and amido modified;

(2) probe synthetic after,, and mix probe dilution with deionized water with sampling liquid sporing solution equal-volume, making final concentration is 75pmols/ μ l, dot matrix places 70% humidity in aldehyde group modified slide surface then, fixes in 48-72 hour under the room temperature condition; The probe design method is characterized in that: whether the feature of design not only can detect hepatitis C virus, and can detect the genotyping information of hepatitis C virus; (3) sample preparation and mark comprise: (a) extracting of serum RNA

5ul sample serum is added on the 0.5ml Eppendorf pipe end, adds 10ulGeneReleaserTM again, mixing, shop one deck paraffin oil is put into the pcr amplification instrument, carries out the gene release procedure: 65 ℃ * 30 seconds, 8 ℃ * 30 seconds, 65 ℃ * 90 seconds, 97 ℃ * 3 minutes, 8 ℃ * 60,65 * 3 minutes, 97 ℃ * 60 seconds, 65 ℃ * 60 seconds, 80 ℃ * 10 minutes; (b) reverse transcription and first round PCR

5ul contains reverse transcriptase primer 10pmol, 70 ℃ of heating of RNasin 20U reverse transcriptase primer liquid 5 minutes, and ice bath adds 10ul and contains overcoat forward primer (P immediately ₃, P ₄) reverse transcription amplification liquid.Smoke tree increases pipe and places the pcr amplification instrument, and 42 ℃ of reverse transcriptions 50 minutes are then carried out amplification program: 94 ℃ * 30 seconds, and 55 ℃ * 30 seconds, 72 ℃ * 40 seconds, 30 circulations; (c) second take turns PCR

3ul first round PCR product and 27ul contain P ₁, P ₂Second of each 10pmol of primer and digoxigenin labeled taken turns the PCR reaction solution and mixed dNTP 0.2Mm, 1 * PCR Buffer, 2mM MgCI2, Taq E enzyme 1.5U] the same PCR for the first time of pcr amplification cycling program; (4) chip hybridization and detection:

Get the PCR product 2 μ l of mark, 98 ℃ of sex change 4mins, then with 20 μ l Easy-Hb hybridization solution mixings, getting 10 μ l drips in the chip surface that has fixed, covered places 42 ℃ of wet boxes to hybridize 25mins, puts then to swing among the washing lotion I and washes 10mins, airing is at last with GenePi 4000B scanning of GenePix Pro chip signal analytical system and analytical results.

4, by described a kind of rapid detection hepatitis C virus and the genotypic chip preparation method thereof of being used for of claim 3, it is characterized in that the probe dot matrix is by the CartesianMicroarray manufacturing system in aldehyde group modified slide surface.

5, by claim 3 described a kind of be used for rapid detection hepatitis C virus and genotypic chip manufacture method thereof with, it is characterized in that described P1, P2, P3, P4 from 5 ' to 3 ' primer sequence be followed successively by 5 '-CACTCGCAAGCACCCTATCAGGCAGT-3, ' 5 '-the corresponding Tm of TCTAGCCATGGCGTTAGTA (C/T) GAGTGT-3 ', 5 '-GCTCATG (G/A) TGCACGGTCTACGAGACCT-3 ' and 5 '-CCCTGTGAGGAACT (A/T) CTGTCTTCACGC-3 ' is followed successively by 71 ℃, 65 ℃, 70 ℃ and 67 ℃.

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