CN1392268A - Detection type gene chip for detecting various peptitis - Google Patents

Abstract

The present invention provides a detection gene chip for diagnosing serveral kinds of hepatitis and new primer with high detection rate for using the gene chip in various subtype amplification. The gene chip includes detection quality controlling system and disease diagnosing system. The chip can be used to detect the virus ites of hepatitis B and hepatitis C and detect hepatitis D, hepatitis E, hepatitis G and TTV simultaneously. It has short detection period, high diagnosis accuracy and low diagnosis cost and may be used in epidemiological investigation and blood detection for identifying hepatitis gene type.

Description

A kind of detection type gene chip that detects multiple hepatitis

Technical field

Technical field involved in the present invention is a kind of hepatitis gene chip, particularly a kind of gene chip that detects the hepatitis sudden change.

Background technology

Biochip mainly is meant by plane Micrometer-Nanometer Processing Technology and supramolecule self-assembling technique, in microanalysis unit and the system that the solid chip surface makes up.Biochip can be many difference in functionality devices: integrate, for example, the pre-treatment of biological sample, the extraction of genetic material, the segmental amplification of specific gene, biological probe array and capillary electrophoresis form whole microfluid system, with realize to compound, protein, nucleic acid, cell and other biological components accurately, fast, the screening or the detection of large information capacity.Gene chip is a most important class biochip, and it the is integrated gene probe of dense arrangement in a large number can be analyzed a large amount of genes at short notice, makes people can promptly read and analyze the program of life.

Biochip has extremely important meaning on biological detection, medical test, drug screening and gene sequencing.For example in biology, along with molecular biological continuous development, the Human Genome Project particularly highly visible is implemented to be exponential growth with the data of acid, protein sequence and structure.And the most challenging work of next century to be exactly the people draw finish after, promptly at the back era gene, how we use a large amount of biomolecules Information servicess in human society, and make medical science, treatment produce basic revolution.In medical science, " the subordinate phase medical science on system, organ, tissue, the cell level " transforms to " on the gene level; DNA-RNA-protein-protein and nucleic acid interaction, and the phase III medical science on they and the environmental interaction level ".This gene diagnosis of carrying out on molecular level and gene platform are treated, and will be familiar with the root that disease produces at all, and will be hopeful basic understanding and treat the major disease that comprises cancer.The fundamental change of these biology, medical science, a mensuration and the analysis that basic prerequisite is a gene order.Can carry out gene sequencing and analysis effectively apace, will have influence on the enforcement of the Human Genome Project, thereby influence further developing of biology, medical science.The method that the tradition gene sequencing is adopted comprises a series of numerous and diverse steps such as chemical reaction, gel electrophoresis, picture processing, these method spended times are longer, and operate complicatedly, especially time-consuming aspect large scale sequencing, and be not suitable for portability and check order fast.Traditional gene order surveying method is being carried out in the improved process, is that the biochip technology of representative arises at the historic moment with the gene chip.Biochip technology is with many discontinuous analytic process related in the life science, and as specimen preparation, chemical reaction and analyzing and testing etc. are by adopting microelectronics, technologies such as micromechanics are integrated in the chip, make it serialization, integrated, microminiaturization and automatization.The maturation of this technology and use and will bring a revolution in medical diagnosis on disease and the life science association areas such as treatment, new drug development, judicial expertise, food and environmental monitoring of next century is though obtain and analyze the means that provide strong for bioinformation.

Biochip (gene chip) is an international research focus in recent years always, and advances just with surprising rapidity.There have been many companies to enter the biochip field in the world, worked out PCR and the integrated biochip of DNA array.These systems prepare a PCR micro reaction pool usually on chip, by controlling the temperature cycle of little reaction, carry out gene amplification, in the gene introduced cross pond after then will increasing, with the hybridization of solid phase micro probe array, detect.Affymetrix company then further makes microfluidic channel to the PCR micro reaction pool.Although the existing report that the round pcr chip detection is integrated is characterized in that whole PCR process carries out in a micro reaction pool.Thereby need to be heated up repeatedly, lower the temperature in the same position of device, this can't be incorporated into the action attitude to PCR and follow the tracks of and quantitative analysis.And the need certain hour that heats up at present, lowers the temperature, prolonged the working hour.

Gene chip (DNA Chip) is the crystallization that biotechnology and microelectronic chip merge, and is a kind of new micro device that is made of the Nucleotide array that is fixed in solid phase carrier (as silicon chip, glass, plastics etc.) surface.It is integrated gene probe carries out the complementation coupling by the base sequence with detected gene, realizes the molecular recognition of nucleotide sequence.Since its fast, trace, use advantage accurately, becoming the automatization medical test instrument of a new generation.The high mutation rate of hepatitis virus causes curative effect of medication not high, and aggravates disease.At the probe of mutator gene, carry out complementation coupling by design with the base sequence of detected gene, can be fast, micro-, detect mutator gene, guiding clinical diagnosis and treatment exactly.U.S. affymetrix company in the 1980s art to the beginning of the nineties, taken the lead in carrying out the research of this respect.1992, the said firm's utilization semi-conductor photograph plate technique was at 1cm ²About slide on the synthetic oligonucleotide fragment of original position, first gene chip in the world has been born. simultaneously, the fluorescent mark of probe, technology such as laser confocal scanning and Computer Analysis are development thereupon also.Nineteen ninety-five, first is that the gene chip (micromatrix) of carrier is born in U.S. Stanford university with glass, and this indicates that biochip technology stepped into the period of broad research and application.The Wu Jian of Southeast China University has successfully developed in male laboratory the synthetic high-density gene chip technology of molecular seal original position, indicates domestic high-density gene chip research and the beginning of using.

Viral hepatitis is China's infection rate and the highest transmissible disease of sickness rate.According to estimates, hepatitis b virus carrier has 1.2 hundred million, wherein has the symptom to account for 1/10, and about 1/4 patient transfers to chronic, and 3% patient is changed posthepatitic cirrhosis into.Caused by viral hepatitis more than 90% in the primary hepatocarcinoma.This disease still lacks the specific treatment method so far, so early diagnosis and state of illness monitoring are just particularly important.

The traditional detection hepatitis B judges whether hepatitis B infection is arranged by two double detections behind the detection five indices, detects the liver function five indices and judges the liver injury degree.Treating hepatitis B genes involved site mutation can't detect, thereby has influenced concrete medication; On the other hand, lack enough evidences for hepatitis B epidemiological survey and epidemiological study thereof, if adopt sequence measurement that multisite mutation is carried out the hepatitis B epidemiological survey, expense height not only, and can't promote at home.

Round pcr mainly is as a kind of selectivity outer-gene amplification method and since through 25～35 take turns the circulation after just can make DNA cloning 10 ⁶Doubly, in scientific research and medical test, be widely used for many years.But, forbidden being used for clinical diagnosis by health ministry from June, 98 because there are defectives such as false positive in PCR.Its major cause has: the first, the labile factor that many experiment conditions in the PCR process (primer design and selection, material mixture ratio, reaction times, temperature, loop cycle) etc. cause cause producing the mistake amplification of PCR; It two is that the follow-up electrophoretic detection of PCR process can be judged the fragment that whether obtains length-specific, and can't determine its concrete sequence; Its three, the PCR reaction product forms the DNA that is contained in the aerosol and very easily form the false positive band at running gel after amplification, thereby causes the unreliability of detected result.

The infectious pathogen diagnosing chip is exactly that characterizing gene fragment (target gene) with pathogenic agent to be measured is fixed in and makes chip on the slide, will be from the patients serum extracting DNA or the RNA that go out pathogenic agent behind the amplification label fluorescent, hybridize with chip, hybridization signal is by scanner scanning, yin and yang attribute is judged in machine analysis as calculated again.The superiority that diagnosing chip is different from other detection meanss is:

(1) test sample is all kinds of Disease-causing gene fragments, has improved detection efficiency;

(2) because of need not organism immune response, can diagnose early, and the testing sample consumption is less;

(3) the diagnosing chip technology is the molecular diagnosis method that DNA hybridization technique and fluorescent mark technology combine, and high sensitivity, specificity and reliability are arranged;

(4) the level of automation height is beneficial to large-scale promotion application.

Aspect medical diagnosis on disease, more existing single disease gene diagnosing chip launch, for example the Affymetrix company of the U.S. has utilized gene chip to carry out the research work of AIDS (HIV), and business-like GeneChipHIVPRT diagnosing chip listing is arranged, and is used for the early diagnosis of AIDS.But the application of chip technology in medical diagnosis on disease still is in the starting stage, and also big area is not promoted,

Reason mainly contains:

(1) biochip technology is a brand-new technology, and is very high to the requirement of the talent, technology, fund, this technology of having had only a fraction of scientific research institution and high-tech company at present on top of.

(2) existing chip can only be diagnosed a kind of disease usually, and the chip detection cost is more expensive, is difficult to replace the conventional sense method at present.

(3) chip technology is particularly in the quality-guarantee of many disease detection with detect and await development and innovation aspect the monitoring.Many medicals diagnosis on disease chip is a kind of polygenic reactive system, therefore traditional pcr amplification primer design is had higher requirement aspect the recall rate; Because many medicals diagnosis on disease chip reactive system complicated, new technical requirements has been proposed also for the detection monitoring of reaction result.

In the various diseases that threatens human health, infectious diseases occupies critical positions always.According to The World Health Organization's 99 annual reports, the whole world amounted to 5,390 ten thousand people's death in 98 years, wherein die from infectious diseases meter 130055 ' people, account for 25% of total number of persons, being equivalent on average per hour just has 1500 people to die from such disease, be only second to and die from 31% of cardiovascular disorder, far above dying from 13% of cancer.This 1,300 ten thousand philtrum has 50% to occur in developing country.Various hepatitis are common disease, the frequently-occurring diseases that threatens human life's health always.

Know that now the most HCV-I types of American-European countries infect, and Asian countries is based on the II type, the III type takes second place.Okomoto reports that Japanese chronic hepatitis C patient and healthy blood donor are mainly the II type and infect, and account for 59.3% and 82.4% respectively, and hemophilia people about 50% infects for the I type, and reason is to use the import of the input U.S. to coagulate Factor IX.WangShi report China Beijing chronic hepatitis C patient 86.2% infects for the II type, and it is 13.8% that the III type infects.And the sick human III type infection in Xinjiang accounts for 50%, illustrates that different shaped HCV has certain area and crowd's distribution characteristics.Different genotype infects and to cause clinical course and interferon therapy reaction also performance is different in addition, and it is heavier to infect clinical symptom as the III type, and have to cause that punishing severely hepatopathy is inclined to: II type (Simmonds 1b) infects the insensitive weak effect of interferon therapy.It is good with effect of interferon that the III type infects (Simononds 2a).

After detecting less than hepatitis B again after identifying liver injury, medical diagnosis requires other hepatitis virus is detected.For multiple hepatitis virus detection method is different, what generally use is that enzyme linked immunoassay detects the proteic antibody of virus-specific, the virus-specific albumen of dependovirus gene translation lags behind the interior infection of body of virus and duplicates, can not reflect real viral survival condition, on the other hand, virus stop to duplicate or intravital scavenging process in, these virus-specific albumen will be survived for some time toward contact, the hysteresis quality of this diagnostic result is brought disadvantageous effect to clinical treatment.To diagnosis new problem has been proposed thus, shortcoming that we detect at enzyme linked immunoassay and PCR detects and deficiency have proposed the use gene chip and have detected multiple hepatitis virus and sudden change thereof, enable directly to detect the kind and the mutation type of hepatitis virus, with guiding clinical treatment.

Blood testing in the blood station, hepatitis virus are the conventional projects of blood testing, but existing detection means be made a definite diagnosis above-mentioned four kinds of diseases by experiment once in can't be at one time, detect the cost height and efficiency ratio is lower.For this reason, development research is a kind of to be used for the gene chip of multiple infectious diseases diagnosis, and the carrying out early diagnosis accurately and efficiently of multiple infectious diseases has been become the task of top priority.

Summary of the invention:

The objective of the invention is to have weak point at present PCR detection method, a kind of detection type gene chip that detects multiple hepatitis is provided, this chip is based on nucleic acid probe and is fixed in the array that forms on the solid substrate, is a kind of detection type biochip.This novel integrated gene chip can be finished PCR process, crossover process, cleaning process on this chip.Its result can be detected the result by gene chip scanning instrument, provides results of hybridization, thereby whether the judgement sample has hepatitis virus, which kind of virus, what sudden change has taken place, and instructs medical treatment and prevention hepatitis to infect according to the sudden change result.Aspect detection, disease transmission monitoring, gene chip of the present invention is propagated vertical transmission, daily life contact transmission, blood and blood product and the route of transmission of sexually transmitted disease (STD) identifies that very good effect is arranged.

Along with the development of Protocols in Molecular Biology, it is found that in recent years in the patient body that many HBV infect to exist different HBV gene strain (being diversity or heterogeneity), and prove that they have confidential relation in the morbidity of propagating with clinical hepatitis.The virus replication level of homophyletic not, immunogenicity is all different with propagated grade, and is therefore also different with the influence that lapses to advancing of disease.Therefore all be of great practical significance in Clinical Laboratory and treatment for hepatitis gene chip of the present invention.

In hepatitis virus detection, diagnosis, disease transmission monitoring, the pathogenic factor rapid detection of viral hepatitis is found out infective virus, the utilization in hepatitis gene sudden change detection and the evaluation.

It is to be divided into three detection level: a. detection viruses to have or not that gene chip of the present invention detects; B. virus is carried out the branch hypotype; C. detect significant sudden change (as resistance, immunologic escape, canceration etc.).

Another object of the present invention is because there is closely related property detection in Gene Mutation and hepatitis B route of transmission, the detection and the evaluation of these type of relevant range gene sudden change subspecies, hypotype.

Principle of design of this gene chip and correlation technique thereof such as Fig. 1.

What following table was enumerated is the mutational site that hepatitis B has clinical meaning, we classify according to last 192 the cited full genes of hepatitis B of GENEBANK (http://www.ncbi.nlm.nih.gov) on the basis of following table, have 6 genotype, chosen on this basis partial-length at the nucleotide sequence of 15～35 bases as the probe (see figure 2), synthetic back fixed slide make the probe that detects hepatitis B in the hepatitis gene chip.

The hepatitis C criteria for classification is ununified as yet, and Simmonds is divided into 1a, 1b, 2a, 2b, 3a, 3b, 4a, 5a, 6a type with hepatitis C, and Okomoto is divided into hepatitis C on types such as I, II, III, IV, V.We classify GENEBANK (http://www.ncbi.nlm.nih.gov) according to above-mentioned somatotype to the full gene of hepatitis C, the difference designing probe, chosen on this basis partial-length at the nucleotide sequence of 15～35 bases as probe, I, II, III, IV, the V that can represent Okomoto respectively be the consensus sequence of a type wherein, synthetic back fixed slide make the probe that detects hepatitis C in the hepatitis gene chip.

Equally hepatitis D, hepatitis E, hepatitis G, TTV are sought its consensus sequence respectively, difference designing probe, the probe of making detection hepatitis C in the hepatitis gene chip of synthetic back fixed slide.

In general, each viral target gene selects to have 2-4 nucleic acid conserved regions sequence, and each saltation zone, genotype and hypotype determining area are according to the probe of diagnosis and evaluation needs selection proper amt, and wherein each saltation zone will have two probes at least, promptly one is wild-type, and one is mutant;

All synthetic probes use point sample instrument will receive the solution of upgrading with point sample damping fluid dissolving back and select the solid phase substrate, and under certain conditions, oligonucleotide probe 5 ' end has group to form covalent linkage with the on-chip chemical functional group of solid phase to be connected.

A kind of detection type gene chip that detects multiple hepatitis comprises (1) multiple detection oligonucleotide probe and quality control oligonucleotide probe, and (2) oligonucleotide probe is connected the probe array that forms on the solid phase substrate by arm molecule with covalent linkage.

So-called oligonucleotide probe be meant can with the polynucleotide of goal gene hybridization, length is in several bases to tens base;

So-called detection oligonucleotide probe be meant the multiple hepatitis of chemosynthesis conserved regions, with the complementary oligonucleotide of saltation zone, genotype and the hypotype of disease-related, the probe of saltation zone comprises wild-type and mutant probe, and the probe of genotype and hypotype determining area comprises several genes type and multiple hypotype probe;

So-called arm molecule is meant the long-chain organic compound with double-active group;

So-called quality control probes comprises two kinds of probes at least, i.e. positive control, negative control; Negative control is used to detect hybridization signal mistake or pollution, and whether normally positive control is used for detecting hybridization;

The end that so-called oligonucleotide probe is fixed as oligonucleotide has a special groups, is fixed on solid substrate when surface, forms covalent linkage with the end group of the arm molecule of finishing.

A kind of detection type gene chip that detects multiple hepatitis, when design, each viral target gene is selected to have 2-4 nucleic acid conserved regions sequence, each saltation zone, genotype and hypotype determining area are selected the probe of proper amt according to diagnosis and evaluation needs, wherein each saltation zone will have two probes at least, promptly one is wild-type, and one is mutant.

A kind of detection type gene chip that detects multiple hepatitis, the solid phase substrate of use comprise slide that several different methods handles, silicon chip, ceramic plate, plastic sheet, nitrocellulose membrane, nylon membrane etc.The solid phase substrate carries out finishing with the long-chain organic compound of double-active group, the long-chain organic compound reagent of double-active group commonly used has: N, N-diethoxy aminopropyltriethoxywerene werene, trishydroxymethyl aminosilane (APTES), glutaraldehyde, poly-lysine can adopt a kind of in them or their two kinds of combinations.

A kind of detection type gene chip that detects multiple hepatitis, the amplimer of many groups be equipped with at the high specific of the infectious diseases virus target gene of the required diagnosis of chip, detection, epidemiological survey, its principle of design must be at the high specific position of people's hepatitis virus target gene for each primer, and every pair of primer extension product usually comprises one or more mutable sites.Simultaneously, each Auele Specific Primer gene segment of all viruses of the same race among the GENEBANK that can increase.

A kind of detection type gene chip that detects multiple hepatitis, the primer mark vitamin H of its use, digoxin, fluorescein, or when gene amplification, add vitamin H, digoxin, fluorescein-labeled dUTP; Hybridization back detection method is to use the DigiTAb of nano gold mark to combine fully, use that nano gold mark affinity element (streptavidin or avidin) fully combines with vitamin H, gene chip scanning instrument scans with digoxin respectively; For the DigiTAb of nano gold mark and nano gold mark affinity element (streptavidin or avidin) but direct viewing or scan with the plain scan instrument.

A kind of detection type gene chip that detects multiple hepatitis, in hepatitis virus detection, diagnosis, disease transmission monitoring, the pathogenic factor rapid detection of viral hepatitis is found out infective virus, the utilization in hepatitis gene sudden change detection and the evaluation.Can be used for the detection of one or more hepatitis viruss, comprise hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis G, TTV and their combination thereof.Can be used for the detection of kind or multiple hepatitis virus sudden change, comprise hepatitis A HAV, hepatitis B HBV, hepatitis C HCV, hepatitis D HDV, hepatitis E HEV, hepatitis G HGV, TTV and their combination thereof.

The gene chip that designs and produces according to this method, the diagnosis, detection, the epidemiological survey that can be used for one or more infectious virus, the pathogenic factor that can be used for the rapid detection viral hepatitis, the clinician finds out that infective virus reaches and the transgenation of disease-related, so that can in time obtain medical treatment, monitor the purpose of the state of an illness.On the other hand, because therefore this type of chip can, have direct directive function to clinical treatment with detecting transgenation.

A kind of detection type gene chip that detects multiple hepatitis, the amplimer of many groups be equipped with at the high specific of the infectious diseases virus target gene of the required diagnosis of chip, detection, epidemiological survey, its principle of design must be at the high specific position of people's hepatitis virus target gene for each primer, and every pair of primer extension product usually comprises one or more mutable sites; Must the increase gene segment of all viruses of the same race among the GENEBANK of each Auele Specific Primer.Advantage of the present invention

(1) test sample is all kinds of Disease-causing gene fragments, has improved detection efficiency;

(2) because of need not organism immune response, can diagnose early, and the testing sample consumption is less;

(3) the diagnosing chip technology is the molecular diagnosis method that DNA hybridization technique and fluorescent mark technology combine, and high sensitivity, specificity and reliability are arranged;

(4) the level of automation height is beneficial to large-scale promotion application.

(5) hepatitis is multiple reason morbidity, and only Causative virus just has eight kinds more than, determine patient's pathogenesis, and ordinary method will be used multiple detection means, and method for gene chip is not only simple and convenient, and required plant and instrument quantity is few.

(6) a kind of detection type gene chip that detects multiple hepatitis, the diagnosis, detection, the epidemiological survey that can be used for one or more infectious virus, the pathogenic factor that can be used for the rapid detection viral hepatitis, the clinician finds out that infective virus reaches and the transgenation of disease-related, so that can in time obtain medical treatment, monitor the purpose of the state of an illness.On the other hand, because therefore this type of chip can, have direct directive function to clinical treatment with detecting transgenation.

Description of drawings

The invention will be further described below with reference to accompanying drawing.

Fig. 1 is the principle of design and the correlation technique synoptic diagram of gene chip

Fig. 2 is a kind of structure that detects the detection type gene chip of multiple hepatitis

Fig. 3 is a part hepatitis B virogene Pareto diagram, therefrom can find out the consensus sequence of hepatitis B, carry out in the GENEBANK relatively again the storehouse, thus the primer sequence that can find the specificity height of hepatitis B various hepatitis viruss can be increased again.

Embodiment

Comprise with reference to 2 one kinds of detection type gene chips that detect multiple hepatitis of accompanying drawing: solid substrate 1, as slide, silicon chip etc.; The solid substrate finishing arm molecule 2, as N, N-oxyethyl group aminopropyltriethoxywerene werene, APTES, glutaraldehyde, arm molecule 2 is to be covalently bound on the solid substrate 1; Behind point sample, amido modified oligonucleotide probe 3 is fixed on the solid substrate of having modified arm molecule, and two points of expression have been fixed different probes among the figure, and oligonucleotide probe 3 is to be covalently bound on the arm molecule 2; Oligonucleotide probe 3 comprises detection oligonucleotide probe and quality control oligonucleotide probe.

Embodiment 1

1, primer sequence:

Wherein HBV primerl sequence is as follows:

5′-GTTTTATCATATTCCTCT-3′

5′-GGTTTTATTAGGGTTCAA-3′

Wherein HBV primer2 sequence is as follows:

5′-GAGGCTGTAGGCATAAAT-3′

5′-GGGCATTTGGTGGTCTG-3′

The HCV amplimer has designed two pairs of primers altogether,

Wherein HCV primerl sequence is as follows:

HCVi：5′-GCCTTGTGGTACTGC-3′

HCVlC：5′-5ACCGCTCGGAAGTC-3′

Wherein HCV primer21 sequence is as follows:

HCV2：5′-GGCTTTACCGGCGAC-3′

HCV2C：5′-GCTCATACCAiGCAC-3′

2, serum sample DNA, RNA extracting

After adding 100 μ l serum room temperatures in the 10 μ l DEPC-ethanolic solns (10% is diethylpyrocarbonate (DEPC), 90% for dehydrated alcohol) and leaving standstill 10 minutes, boiling water bath is after 10 minutes, 65 ℃ of oven dry of pipe lid 15 minutes, centrifugal 5 minutes (12000 rev/mins, centrifugal 10 minutes), standby.

3, reverse transcription prepares template DNA

In the sedimentary eppendorf pipe of DNA, RNA that step 2 preparation contains, add following reagent successively:

Milli-Q H2O (ultrapure water) 11 μ l, 15 μ mol/LHCV, HCV, reverse transcription primer 2 μ l and abundant dissolution precipitation, the mixing sample centrifugally in short-term concentrates on solution to manage at the end, place insulation 5min in 70 ℃ of water-baths.After eppendorf is placed 1min on ice, add following reagent: 0.1mol/1DTT (dithiothreitol (DTT)) 2 μ l, reverse transcription damping fluid (buffer) 4 μ l, 10mmol/1dNTPS 1 μ l, centrifugally in short-term solution is concentrated on manage at the end.Place in 42 ℃ of water-baths and be incubated 2 minutes.Adding 0.5 μ l SuperScript ThermoScript II in this eppendorf pipe centrifugally in short-term concentrates on solution to manage at the end.Place in 42 ℃ of water-baths and be incubated 20 minutes.In water-bath, take out the eppendorf pipe, standby.

4, draw the transcription product of preparation in the 5pl step 2, and add following reagent successively:

ddH ₂O?28.5μl

10×PCR?Buffer?5μl

25nunol/1?MgCl2?4μl

10mmol/1 dNTP mixture 1 μ l

1mmol/1?dTTP?1μl

1mmol/1?spectrum-red-dUTP?0.2μl

15pmol/1 HBV, HCV primer mixture 1 μ l

Taq enzyme 0.5 μ l

Centrifugally in short-term solution is concentrated on manage at the end, 95 ℃ of 30sec are carried out in the PCR reaction behind 95 ℃ of sex change 3min in the PCR instrument, 55 ℃ of 30sec, and 72 ℃ of 30sec circulations 30 times are extended at 72 ℃ of 5min again.At last 4 ℃ of standbies.

5, the product of step 4 is put on the detection chip after adding hybridization solution, and covered is incubated 20 minutes down at 37 ℃.

6, cleaned hybridization hybrid chip respectively each 5 minutes with hybridization scavenging solution I and II.Use N ₂Dry up.

7, with scanligh scanner scanning gene chip.Imagene software analysis results of hybridization.Scanning result shows over against shining fluorescent signal; Negative contrast does not have fluorescent signal, judges that according to having or not of signal testing sample is the feminine gender or the positive, and hence one can see that, and whether tested serum infect has with the virus of inspection and whether have significant transgenation, also can detect gene type wherein simultaneously.

Embodiment 2

1, the following probe of design is used for virus is carried out the detection of branch hypotype

	Rating	?Seq.No	?Length	???Tm	??GC％	????Sequence
							????1 ????1’	??86	???505	??16	??52.7	??62.5	??CACGGGACCA?TGCAAG ??CACGGGACCA?TGCCGG

????2 ????2’	????88	????507	????17	??53.7	??58.8	??CGGGACCATG?CAAGACT ??CGGGACCATG?CCGGACT
							????3 ????3’	????100	????627	????20	??55.6	??45.0	??TCGCAAAATA?CCTATGGGAG ??TCGCAAGATA?CCTATGGGAG
????4 ????4’	????100	????629	????21	??56.7	??47.6	??GCAAAATACC?TATGGGAGTG?G ??GCAAGATACC?TATGGGAGTG?G

Hepatitis B has the mutational site of clinical meaning

Numbering	Nucleotide position	The mutational site	Amino acid position	Amino acid mutation	The clinical meaning of sudden change
						???1	????525	????G-A	S district 124	??Cys→Tyr	HBV infects HBsAg (-) after the HBIG prevention liver transplantation
???2	????527-529	????ACA→GCA ????ACA→GCG	S district 126	??Thr→Ala ??Ile→Ser	Immune evasion HBsAg (-) HBsAb (-)
						???3	????546	????C→T	S district 131	??Thr→Ile	HBV infects HBsAg (-) after the HBIG prevention liver transplantation
???4	????552	????T→C	S district 133	??Met→Thr	Immune evasion, HBsAg (-)
						???5	????569	????T→A	S district 139	??Cys→ATG	?HBsAg(+)
???6	????575	????A→G	S district 141	??Lys→Glu	?HBsAg(+) ?HBsAb(+)
						???7	????587	????G→A	S district 145	??Gly→Arg	HBV infects HBsAg (+), HBsAb (+) after immune evasion, the HBIG prevention liver transplantation
???8	????624	????C→A	S district 157	L-Ala → aspartic acid	Lamivudine opposing HBV strain is influential to the HBsAg antigenicity
						???9	????739-740	????AT→TG	S district 195	Methionine(Met) → tryptophane	Lamivudine opposing HBV strain
???10	????1653	????C→T	The X district		Fulminant hepatitis and chronic hepatitis acute failure
						???11	????1762/1764	????A→T ????G→A	The X district	Basic promotor (BCP) sudden change	Fulminant hepatitis and the death of chronic hepatitis acute failure
???12	????1862	????G→T	Preceding C district 17	A word used in person's names propylhomoserin → phenylalanine	The negative hepatitis gravis of HBeAg
						???13	????1896	????G→A	Preceding C district 28	Tryptophane → terminator codon
???14	????1899	????G→A	Preceding C district 29	Tryptophane → terminator codon	The interferon-alpha treatment drug-induced variation in back, fulminant hepatitis
						???15	????2078	????C→G	C district 60		Adw fulminant hepatitis and chronic hepatitis
???16	????2157-2158	????TC→GG	C district 87	Serine → sweet	The adr fulminant hepatitis and

				Propylhomoserin	Chronic hepatitis
						????17	????2189	????A→C	C district 98	Isoleucine → leucine	Chronic viral hepatitis B
????18	????2798	????A→C	P district 163	Threonine → proline(Pro)	The synthetic of viral DNA is blocked

Being used for that according to the following probe of meaningful sudden change Position Design in the last table virus is carried out mutation type detects

??S?gene	Detect sudden change	???Seq.No	??Length	??Tm	??GC％	????Sequence
							????1 ????1’ ????2’ ????2”	??1、2	????518	????20	??55.6	??50.6	??AAGACTTGCA?CAGCTCCTGC ??AAGACTTACA?CAGCTCCTGC ??AAGACTTGCG?CAGCTCCTGC ??AAGACTTGCG?CGGCTCCTGC
????3 ????3’ ????4’	??3、4	????539	????20	??54.2	??45.4	??CAAGGAACCT?CTATGTTTCC ??CAAGGAATCT?CTATGTTTCC ??CAAGGAACCT?CTACGTTTCC
							????5 ????5’ ????6’	??5、6	????562	????21	??55.7	??41.0	??ATGTTGCTGT?ACAAAACCTA?C ??ATGTTGCAGT?ACAAAACCTA?C ??ATGTTGCTGT?ACAGAACCTA?C
????7’	????7	????579	????18	??54.4	??53.1	??CTACGGACAG?AAACTGCA
							????8’	????8	????617	????19	??54.0	??44.5	??TCTTGGGATT?TCGCAAAAT
????9’	????9	????731	????20	??54.7	??41.3	??TCAGTTATTG?GGATGATGTG
							??X?gene		???Seq.No	??Length	??Tm	??GC％	????Sequence
????10’	????10	????1641	????22	??55.0	??50.0	??CCCAAGGTCT?TGTATAAGAG?AA
							????11’	????11	????1748	????23	??55.0	??43.1	??GGAGGTTAGG?TTAATGATCT?TTG
??Precore		???Seq.No	??Length	??Tm	??GC％	????Sequence
							????12’	????12	????1852	????21	??53.5	??47.6	??ATGTCCTACT?TTTCAAGCCT?C
????13’ ????14’	????13	????1889	????17	??55.0	??52.9	??TGGCTTTAGA?GCATGGA ??TGGCTTTGGA?ACATGGA
							??C?gene		???Seq.No	??Length	??Tm	??GC％	????Sequence
????15’	????14	????2065	????20	??53.0	??45.0	??CAGGCAAGCT?ATTGTGTGTT
							????16’	????15	????2149	????24	??53.6	??41.7	??CTTAGTAGGG?AGCTATGTCA?ACGT
????17 ????17’	????16	????2179	????19	??52.7	??42.1	??GGGCCTAAAA?TTCAGACAA ??GGGCCTAAAC?TTCAGACAA
							??P?gene		???Seq.No	??Length	??Tm	??GC％	????Sequence
????18’	????17	????2798	????20	??56.1	??58.0	??CCACACGTCG?CGCCTCATTT

2, above-mentioned probe is synthetic and at 5 ' end mark amino, with the dilution of point sample damping fluid, with point sample instrument with sample spot to the slide of having modified the arm molecule, fixing.

Serum sample DNA, RNA extracting

After adding 100 μ l serum room temperatures in the 10 μ lDEPC-ethanolic solns (10% is diethylpyrocarbonate (DEPC), 90% for dehydrated alcohol) and leaving standstill 10 minutes, boiling water bath is after 10 minutes, 65 ℃ of oven dry of pipe lid 15 minutes, centrifugal 5 minutes (12000 rev/mins, centrifugal 10 minutes), standby.

3, reverse transcription prepares template DNA

In the sedimentary eppendorf pipe of DNA, RNA that step 2 preparation contains, add following reagent successively:

Milli-Q H2O (ultrapure water) 11 μ l, 15 μ mol/LHCV, HCV, reverse transcription primer 2 μ l and abundant dissolution precipitation, the mixing sample centrifugally in short-term concentrates on solution to manage at the end, place insulation 5min in 70 ℃ of water-baths.After eppendorf is placed 1min on ice, add following reagent: 0.1 mol/1DTT (dithiothreitol (DTT)), 2 μ l, reverse transcription damping fluid (buffer) 4 μ l, 10mmol/1dNTPS 1 μ l centrifugally in short-term concentrate on solution to manage at the end.Place in 42 ℃ of water-baths and be incubated 2 minutes.Adding 0.5 μ l SuperScript ThermoScript II in this eppendorf pipe centrifugally in short-term concentrates on solution to manage at the end.Place in 42 ℃ of water-baths and be incubated 20 minutes.In water-bath, take out the eppendorf pipe, standby.

4, draw the transcription product of preparation in the 5pl step 2, and add following reagent successively:

ddH2O?28.5μl

10×PCR?Buffer?5μl

25nunol/1?MgCl2?4μl

10mmol/1 dNTP mixture 1 μ l

1mmol/l?dTTP?1μl

1mmol/l?spectrum-red-dUTP?0.2μl

15pmol/1 HBV, HCV primer mixture 1 μ l (only adding reverse primer)

HOT START Taq enzyme 0.5 μ l

Centrifugally in short-term solution is concentrated on manage at the end.

5, the mixed solution in the step 4 is added in chip surface, put a GENE FRAME, add a cover glass, place MJ chip PCR instrument to carry out the PCR reaction chip, carry out 95 ℃ of 30sec behind 95 ℃ of sex change 5min, 55 ℃ of 30sec, 72 ℃ of 30sec circulate 30 times, extend at 72 ℃ of 5min again.At last 4 ℃ of standbies.

6, cleaned hybridization hybrid chip respectively each 5 minutes with hybridization scavenging solution I and II.Use N ₂Dry up.

7, with scanligh scanner scanning gene chip.Imagene software analysis results of hybridization.Scanning result shows over against shining fluorescent signal; Negative contrast does not have fluorescent signal, judges that according to having or not of signal testing sample is the feminine gender or the positive, and hence one can see that, and whether tested serum infect has with the virus of inspection and whether have significant transgenation, also can detect gene type wherein simultaneously.

Embodiment 3

1, designing probe is used for viruses such as HBV, HCV, HDV, HGV, TTV are carried out the virus detection; Designing probe is used for HBV, HCV branch hypotype are detected; Designing following probe is used for HBV virus is carried out the mutation type detection.

2, above-mentioned probe is synthetic and at 5 ' end mark amino, with the dilution of point sample damping fluid, with point sample instrument with sample spot to the slide of having modified the arm molecule, fixing.

Serum sample DNA, RNA extracting

After adding 100 μ l serum room temperatures in the 10 μ l DEPC-ethanolic solns (10% is diethylpyrocarbonate (DEPC), 90% for dehydrated alcohol) and leaving standstill 10 minutes, boiling water bath is after 10 minutes, 65 ℃ of oven dry of pipe lid 15 minutes, centrifugal 5 minutes (12000 rev/mins, centrifugal 10 minutes), standby.

3, reverse transcription prepares template DNA

In the sedimentary eppendorf pipe of DNA, RNA that step 2 preparation contains, add following reagent successively:

Milli-Q H2O (ultrapure water) 11 μ l, 15 μ mol/L HCV, HDV, HGV, TTV reverse transcription primer 2 μ l and abundant dissolution precipitation, the mixing sample centrifugally in short-term concentrates on solution to manage at the end, place insulation 5min in 70 ℃ of water-baths.After eppendorf is placed 1min on ice, add following reagent: 0.1mol/lDTT (dithiothreitol (DTT)) 2 μ l, reverse transcription damping fluid (buffer) 4 μ l, 10mmol/l dNTPS 1 μ l, centrifugally in short-term solution is concentrated on manage at the end.Place in 42 ℃ of water-baths and be incubated 2 minutes.Adding 0.5 μ l SuperScript ThermoScript II in this eppendorf pipe centrifugally in short-term concentrates on solution to manage at the end.Place in 42 ℃ of water-baths and be incubated 20 minutes.In water-bath, take out the eppendorf pipe, standby.

4, draw the transcription product of preparation in the 5pl step 2, and add following reagent successively:

ddH2O?28.5μl

10×PCR?Buffer?5μl

25?nunol/1?MgCl2?4μl

10 mmol/l dNTP mixtures, 1 μ l

1?mmol/l?dTTP?1μl

1?mmol/l?spectrum-red-dUTP?0.2μl

15pmol/l HBV, HCV primer mixture 1 μ l (only adding reverse primer)

HOT START Taq enzyme 0.5 μ l

Centrifugally in short-term solution is concentrated on manage at the end.

5, the mixed solution in the step 4 is added in chip surface, put a GENE FRAME, add a cover glass, place MJ chip PCR instrument to carry out the PCR reaction chip, carry out 95 ℃ of 30 sec behind 95 ℃ of sex change 5 min, 55 ℃ of 30 sec, 72 ℃ of 30 sec circulates 30 times, extends at 72 ℃ of 5 min again.At last 4 ℃ of standbies.

6, cleaned hybridization hybrid chip respectively each 5 minutes with hybridization scavenging solution I and II.Use N ₂Dry up.

7, with scanligh scanner scanning gene chip.Imagene software analysis results of hybridization.Scanning result shows over against shining fluorescent signal; Negative contrast does not have fluorescent signal, judges that according to having or not of signal testing sample is the feminine gender or the positive, and hence one can see that, and whether tested serum infect has with the virus of inspection and whether have significant transgenation, also can detect gene type wherein simultaneously.

Claims (10)

1. detection type gene chip that detects multiple hepatitis, it is characterized in that chip comprises (1) multiple detection oligonucleotide probe and quality control oligonucleotide probe, (2) oligonucleotide probe is connected the probe array that forms on the solid phase substrate by arm molecule with covalent linkage.

So-called oligonucleotide probe be meant can with the polynucleotide of goal gene hybridization, length is in several bases to tens base;

So-called detection oligonucleotide probe be meant the multiple hepatitis of chemosynthesis conserved regions, with the complementary oligonucleotide of saltation zone, genotype and the hypotype of disease-related, the probe of saltation zone comprises wild-type and mutant probe, and the probe of genotype and hypotype determining area comprises several genes type and multiple hypotype probe;

So-called arm molecule is meant the long-chain organic compound with double-active group;

So-called quality control probes comprises two kinds of probes at least, i.e. positive control, negative control; Negative control is used to detect hybridization signal mistake or pollution, and whether normally positive control is used for detecting hybridization;

The end that so-called oligonucleotide probe is fixed as oligonucleotide has a special groups, is fixed on solid substrate when surface, forms covalent linkage with the end group of the arm molecule of finishing.

2. a kind of detection type gene chip that detects multiple hepatitis according to claim 1, it is characterized in that: each viral target gene selects to have 2-4 nucleic acid conserved regions sequence, each saltation zone, genotype and hypotype determining area are selected the probe of proper amt according to diagnosis and evaluation needs, wherein each saltation zone will have two probes at least, promptly one is wild-type, and one is mutant.

3. a kind of detection type gene chip that detects multiple hepatitis according to claim 1 and 2 is characterized in that: the solid phase substrate comprises slide that several different methods handles, silicon chip, ceramic plate, plastic sheet, nitrocellulose membrane, nylon membrane etc.

4. a kind of detection type gene chip that detects multiple hepatitis according to claim 1 and 2, it is characterized in that: be equipped with the amplimer of many groups at the high specific of the infectious diseases virus target gene of the required diagnosis of chip, detection, epidemiological survey, its principle of design must be at the high specific position of people's hepatitis virus target gene for each primer, and every pair of primer extension product usually comprises one or more mutable sites.

5. a kind of detection type gene chip that detects multiple hepatitis according to claim 1 and 2 is characterized in that: can the increase gene segment of all viruses of the same race among the GENEBANK of each Auele Specific Primer.

6. a kind of detection type gene chip that detects multiple hepatitis according to claim 1 and 2, it is characterized in that: the solid phase substrate carries out finishing with the long-chain organic compound of double-active group, the long-chain organic compound reagent of double-active group commonly used has: N, N-diethoxy aminopropyltriethoxywerene werene, trishydroxymethyl aminosilane (APTES), glutaraldehyde, poly-lysine can adopt a kind of in them or their two kinds of combinations.

7. a kind of detection type gene chip that detects multiple hepatitis according to claim 1 and 2 is characterized in that: but primer mark vitamin H, digoxin, fluorescein, or when gene amplification, add vitamin H, digoxin, fluorescein-labeled dUTP; Hybridization back detection method is to use the DigiTAb of nano gold mark to combine fully, use that nano gold mark affinity element (streptavidin or avidin) fully combines with vitamin H, gene chip scanning instrument scans with digoxin respectively; For the DigiTAb of nano gold mark and nano gold mark affinity element (streptavidin or avidin) but direct viewing or scan with the plain scan instrument.

8. claim 1 or 2 described a kind of detection type gene chips that detect multiple hepatitis, in hepatitis virus detection, diagnosis, disease transmission monitoring, the pathogenic factor rapid detection of viral hepatitis is found out infective virus, the utilization in hepatitis gene sudden change detection and the evaluation.

9. according to claim 1 or 8 described a kind of detection type gene chips that detect multiple hepatitis, it is characterized in that: can be used for the detection of one or more hepatitis viruss, comprise hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis G, TTV and their combination thereof.

10. according to claim 1 or 8 described a kind of detection type gene chips that detect multiple hepatitis, it is characterized in that: can be used for the detection of kind or multiple hepatitis virus sudden change, comprise hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis G, TTV and their combination thereof.

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