CN108118083A - Multi-biotin signal amplification method - Google Patents
Multi-biotin signal amplification method Download PDFInfo
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- CN108118083A CN108118083A CN201611086876.8A CN201611086876A CN108118083A CN 108118083 A CN108118083 A CN 108118083A CN 201611086876 A CN201611086876 A CN 201611086876A CN 108118083 A CN108118083 A CN 108118083A
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/682—Signal amplification
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
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- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
- C12Q1/706—Specific hybridization probes for hepatitis
- C12Q1/707—Specific hybridization probes for hepatitis non-A, non-B Hepatitis, excluding hepatitis D
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Abstract
A kind of multi-biotin signal amplification method, it is amplified to be promoted the sensitivity of detection of nucleic acids to signal to be detected by the connection of a multi-biotin molecule, which includes the single stranded nucleic acid molecule by hybridizing identification attachment molecule and the carrier molecule for biotin labeling.So as to which a target molecule can be connected with connection molecule, carrier molecule and multiple biotins and corresponding label and amplified the signal of detection.Multiple attachment molecules in the different zones connection of target molecule can obtain the amplification effect of bigger.
Description
Technical field
Detection the present invention relates to the detection of living matter more particularly to vital signs substance.
Background technology
Vital signs substance includes two major class of DNA and RNA.
By taking DNA material as an example, hepatitis B is a kind of hepatic infection as caused by hepatitis B, to body potential threat compared with
Greatly, it is the virus hepatitis type of a kind of main health problem and most serious in the world.Hepatitis B is in Chinese and other Asia
Area is popular, is estimated to be 2,000,000,000 people and is subject to hepatitis B virus infection.Although Prevalent district takes plan, vaccine successfully makes hepatitis B table
Face antigen (HBsAg, the serologic marker of chronic infection) popularity degree declines, and still has 3.5 hundred million virus carrier, wherein 25%
Hepatopathy, hepatic sclerosis and primary hepatoma (HCC) will be developed into, 600,000 people is estimated to be every year and dies of the anxious slow of hepatitis B initiation
Property disease.The Chinese annual direct medical expenses of HBV are about more than 26,000,000,000 RMB.
Marker in blood can be used for hepatitis B infected diagnosis and the antidiastole of acute and chronic infection, these marker bags
Include the antigen and antibody of hepatitis B.There are three types of common antigen --- surface antigen (HBsAg), existing HBV senses for hepatitis B
Dye diagnosis is based on the immunology detection to surface antigen, core antigen (HBcAg) and e antigens (HBeAg) and its corresponding antibody.
Although these detections can be used for carrier's primary dcreening operation, the detection of infectious virus in serum cannot be used for.
HBV-DNA is the most direct mark of virus replication.HBV is small DNA virus, and core shell contains the portion of a 3.2Kb
Divide duplicate factor group, replicate and pass through RNA mediations.It is to differentiate the important work whether virus replicates to measure serum HBV-DNA
Tool detects that the HBV-DNA marks person virus in blood specimen is active and replicates.It is soon after oxyhepatitis, HBV-DNA infection
It is existing, it then disappears after removing infection.In chronic hepatitis B, HBV-DNA levels usually persistently increase for many years, when immune system control is viral
Then decline.For chronic hepatitis B, some HBsAg are positive, anti-HBe is positive and patient that HBV is actively replicated, are detected using PCR special
It is not useful.
There is the patient that 5-10% carries out liver transfer operation to suffer from the relevant chronic and fulminant hepatopathys of HBV.HBV infects again
Due to deriving from circulating, the transplanting of the HBV virions of position or the two directly infects again outside liver.There is no preventive test,
The risk that HBV infects again is almost 80%.Recidivity infection can cause graft failure, transplant again or dead.Therefore liver transfer operation
Follow-up HBV infection is most important to long term survival.This just needs more sensitive HBV-DNA to detect.
Antiviral therapy is control and the unique selection for preventing chronic hepatitis B progression of disease.Verified nucleoside analog exists
It is effective to control disease process, but drug resistance limits long-term effect.Lamivudine is that first approval is used for
The nucleoside analog of disease treatment.The annual incidence of lamivudine drug resistance is 14-24%, and the incidence of 4-5 is 70%.
Due to the generation of drug resistance strain, intactly monitor patient and become more and more important, it is anti-that various mutations occur mainly in HBV polymerases
The active site in transcriptase area, YMDD.Have two it is main mutation it is related with lamivudine drug resistance：RtM204V and
RtM204I, others mutation have rtL80V, rtI169T, rtV173L, rtL180M, rt181T, rtT184S, rtM240V/I/S
And rtQ215S.
HBV Resistance detections based on PCR method at present, but this have two it is potential the problem of, being primarily due to DNA pollution can
It can cause false positive, furthermore it is very limited amount of to detect various mutations simultaneously with PCR.
PCR is a kind of virus genomic method of the quick detection of direct height, but caused by pollution the risk of false positive and
Difficulty on development quantitative detecting method may prevent its application.Further, since the incomplete denaturation of suboptimum reaction condition and DNA
It can cause false negative, the mistake in terms of sample collection and/or processing may also cause problem.
By taking RNA substances as an example, Hepatitis C Virus (HCV) is another main cause of the related morbidity of liver and death,
Chronic infection caused by there are 1.7 hundred million HCV in the whole world accounts for the 3% of world population.Its long-term serious clinical disease risk is hepatic sclerosis
And liver cancer.China has 300,000 people to die of acute and chronic hepatitis B and hepatitis every year, and identical with HBV, HCV infection can cause acute and chronic third
The hepatocellular carcinoma (HCC) of liver, hepatic sclerosis and primary.Researches show that compared with HBV, HCV and HCC relations are closer, 80%HCC
Caused by HCV, caused by HBV, be 15%.It is that HCV infection is mediated with fatty liver, liver that HCV infection is visibly different with HBV
Diabetes, B cell lymphoma it is related.The Chinese annual direct medical expenses of HCV are about in 117 hundred million to 216 hundred million RMB.
HCV is single-stranded RNA virus, there is quick spontaneous mutation, and Frequency Estimation is that annual each nucleotide is 1022
Secondary mutation, cause in the world many separated virus sequences it is different larger, this so that vaccine development is highly difficult, therefore efficient diagnosis
HCV is most important to the propagation for preventing hepatitis, this will save medical resource on long terms, reduces the medical expenses of HCV.Based on one
Fixed gene difference and similitude, HCV are segmented into 6 main genotype, and 1,2,3,4,5 and 6, it is distributed in different states
Family, 2 types are the most common types of China, and Europe and the U.S. are 1 types.Genotype information is very important HCV therapy, can
Predicted treatment effect, such as with glycol interferon Ribavirin is added to treat 2 types or 3 types 24 weeks, 1 type is 48 weeks.
30% post-transfusion hepatitis is due to hepatitis C in China, and blood transfusion is the main source of HCV infection, big at present
The inspection of most hospitals anti-HCV intrusions not before examination.Many countries introduce " guide " of HCV therapy, and China will also push away
Row " Chinese hepatitis C prevention guideline ", this, which will improve, plays positive effect in existing diagnosis of hepatitis C.Hepatitis C
Early diagnosis is to preventing being propagated further among people at highest risk and clinician from making quickly treating determine it is to pass
Important, because verified quick treatment is very effective acute hepatitis.
The infection of Hepatitis C Virus can be determined by detecting in blood HCV antibody.HCV-Ab IgG C-100 antibody is put
Exempt from detection method and ELISA detection method be hepatitis C Main Diagnosis method, this is there are many shortcoming, and first, anti-C100 resists
Body appearance is later, and the window phase of 8 weeks cannot be detected after acute infection.It is after transfusion that hepatitis patient, which has half, after blood transfusion
The 4-6 months first appear the serological conversion of anti-C-100, be accordingly used in oxyhepatitis routine experiment diagnosis just it is improper, second is not
Sensitivity, a small amount of hepatitis patient can't detect C-100 antibody, and the 3rd is special, the patients of some chronic liver diseases due to itself
Immune factor causes false positive, and since the appearance of anti-HCV core antibody is more early, two generations detection HCV antigen/antibody combination detection reagent is based on
The C-22-C albumen in HCV C areas and non-structural district NS3 encoding proteins C-33-3 and C-100-3, this causes detection efficiency to improve
25~30%, the primary detection phase is made to shift to an earlier date 16-42 days.
Due to many factors, including antigenic mutation, more genotype, longer window phase etc., individual antibody test
Or antigen detection is not reliable method in the secure context for examination of donating blood, it is inefficient in the HCV infection diagnosis of early stage.It is existing
It is diagnosis virus itself and its a kind of more direct more reliable approach replicated in HCV-RNA detections.Due in HCV infection person's blood
HCV amounts are limited, this is difficult to not expanded direct cross to detect HCV-RNA.HCV is RNA virus, before PCR amplification,
It needs RNA being first converted into cDNA, with the primer of the conserved regions design of 5 ' noncoding regions come reverse transcription and amplification, amplified production electricity
Swimming observation, method are very sensitive.HCV-RNA/PCR can be used to measure the HCV-RNA in liver and serum, can (the sense in as little as 1-2 weeks
After dye) detect HCV.Since liver and serum HCV-RNA are compared with the morning that antibody occurs, many HCV infection persons do not occur HCV-Ab IgG blood also
During clear conversion, HCV-RNA can be detected in liver and serum.During the HCV-RNA positives, show virus replication；HCV-RNA is cloudy
During property, show virus sweep.Therefore RT-PCR is available for the early diagnosis of HCV, screening blood supply and indication hepatitis prognosis.But
The major defect of the RT-PCR detections of HCV is to need to prepare RNA and reverse transcription cDNA, additionally needs certain technology,
Reappearance is limited, it is necessary to which specific instrument and equipment, there is contaminated danger.
The content of the invention
The technical problem to be solved in the present invention is to overcome above-mentioned the deficiencies in the prior art and propose a kind of inspection of high sensitivity
Survey technology.The technology of the present invention is amplified by signal rather than the template amplification technology of based on PCR realizes that the sensitive of nucleic acid is detected.
Because not must template amplification, so as to the pollution problem for avoiding PCR intrinsic.More importantly PCR is only used for DNA cloning.For RNA
The mRNA labels of molecule such as HCV RNA virus and many, PCR just need that RNA is converted into cDNA before amplification.Because
The technology of the present invention can directly detect RNA molecule, not must reverse transcription and the RNA pre-separations that are carried out to transcribe, therefore
Greatly simplify operating procedure.In addition, unlike PCR the present invention technology have broader linear detection range and more preferably
Accuracy, when detection is not required special instrument and equipment, the preliminary making dyestuff that when quantitative determination need not be expensive, and detection is both simple
It is single and efficient.
The present invention solve above-mentioned technical problem the technical solution adopted is that, manufacture and design a kind of multi-biotin signal amplification side
Method is amplified signal to be detected by the connection of a multi-biotin molecule to promote the sensitivity of detection of nucleic acids, this is mostly raw
Biotin includes the single strand dna by hybridizing identification attachment molecule and the carrier molecule for biotin labeling.
The carrier molecule is DNA, RNA, oligonucleotides, polynucleotides or polymer.
The carrier molecule is single-stranded or double-stranded.
Biotin on the carrier molecule is the biotin by enzyme reaction or chemical reaction hybridization mark.
The biotin labeling makes at least two biotins covalently or non-covalently be connected to the carrier molecule.
The attachment molecule is DNA, RNA or oligonucleotides.
The attachment molecule is the part with one or more synthetic molecules of target hybridization.
The target molecule is to capture on microwell plate or in other solid phases.
In one embodiment, which is HBV-DNA.
In another embodiment, which is HCV-RNA.
Compared with the existing technology, multi-biotin signal amplification method of the invention can greatly improve the signal of detection
Detection sensitivity, and exempt various technological deficiencies present in PCR.
Description of the drawings
Fig. 1 is the basic principle of the multi-biotin signal amplification method of the present invention.
Fig. 2 is the operation principle of the multi-biotin signal amplification method of the present invention.
Specific embodiment
It is described in further detail below in conjunction with the most preferred embodiment shown in each attached drawing.
As shown in Figure 1, the basic principle of the multi-biotin signal amplification method of the present invention is that the target molecule 1 can capture
On to microwell plate 6 or in other solid phases, which can be with a connection molecule 2, multi-biotin molecules 4 and a phases
The label 5 answered connects, to realize the detection of the target molecule 1.
As shown in Fig. 2, the operation principle of the multi-biotin signal amplification method of the present invention is that the target molecule 1 can capture
On to microwell plate 6 or in other solid phases, the target molecule 1 can with connection molecule 2, carrier molecule 3, be carried on each carrier molecule
Biotin molecule 4 and corresponding label 5 on 3 connect, to realize the detection of the signal of the target molecule 1 amplification.It needs
Bright, the number in the diagram, operation principle only to illustrate the invention is used, and does not form the restriction to the method for the present invention,
Wherein,
The attachment molecule 2 can be DNA, RNA or oligonucleotides, be the one or more conjunctions hybridized with target molecule 1
Into a part for molecule.
The carrier molecule 3 be DNA, RNA, oligonucleotides, polynucleotides or polymer, can be it is single-stranded or double-stranded,
Can be by biotin 4 in enzyme reaction or chemical reaction hybridization mark, which makes at least two biotins 4 covalently
Or it is not covalently linked to the carrier 3.
So as to which target molecule 1 can be to multiple connection molecules 2, multiple carrier molecules 3 and multiple biotins 4 and corresponding
Label 5 connect and the signal of detection made to obtain amplification this technology and is known as multi-biotin signal amplification method (MBSA).
By taking the detection of HBV-DNA as an example, multi-biotin signal amplification method of the invention by amplifying report signal, without
It is amplification target sequence, therefore avoids and be difficult to the error broken away from caused by during extracting and expanding target sequence, in addition, institute
Have the drug-fast mutant sites of known HBV drugs, measure that can be equal based on MBSA targets probe sequence, the technology of MBSA and
Practicability feature contributes to conventional use clinically.Specimen preparation procedure is not needed for serum and plasma specimen, for having
Potential pollution sample, must not special processing procedure.Can nucleic acid extraction be omitted with Accurate Determining virus load using MBSA technologies
Tedious steps reduce the possibility of cross contamination.
The multi-biotin signal amplification method of the present invention is similar to sandwich ELISA method, in this method, the serum from patient
It can be fed directly to after DNA denaturation in detection carrier, such as 96 hole microwell plates, HBV-DNA is by few core pre-coated in microwell plate
Thuja acid specifically captures, and after capture, HBV-DNA molecules are incubated with the oligonucleotides in detection sample, are divided afterwards with multi-biotin
Son is incubated, and so as to which multiple rather than single streptomysin and element-horseradish enzyme can be combined with the HBVDNA molecules of capture, forms detection
The amplification of signal, therefore can high quick detection HBV-DNA.
By taking the detection of HCV-RNA as an example, multi-biotin signal amplification method of the invention is surveyed by amplifying report signal
Determine HCV-RNA levels rather than amplification target sequence, therefore avoid and be difficult to caused by during extracting and expanding target sequence
The error broken away from.The target probe of MBSA Technology designs can quantify all known HCV genotype on an equal basis, therefore this method can
With the equal all rna transcription bodies for quantitatively including 6 major HCV genotypes sequences.The technology of MBSA and practicability feature
Contribute to conventional use clinically.Specimen preparation procedure is not needed for serum and plasma specimen, for there is potential pollution mark
This, must not special processing procedure.The tedious steps of nucleic acid extraction with Accurate Determining virus load, can be omitted using MBSA technologies,
Reduce the possibility of cross contamination.
Similar to sandwich ELISA method, the serum from patient becomes the multi-biotin signal amplification method of the present invention in nucleic acid
It can be fed directly in 96 orifice plates after property, HCV-RNA is specifically captured by oligonucleotides pre-coated in microwell plate, after capture,
HCV-RNA molecules are incubated with the oligonucleotides in detection sample, are incubated afterwards with multi-biotin molecule, multiple rather than single chain
Mycin and element-horseradish enzyme are combined with the HCV-RNA molecules captured, therefore can high quick detection HCV-RNA.
To sum up, multi-biotin signal amplification method method of the invention generally comprises three incubation steps：
1st, sample and detection oligonucleotides incubate；
2nd, it is incubated with multi-biotin molecule；
3rd, incubated with streptomysin and element-horseradish enzyme.
As it can be seen that the multi-biotin signal amplification method of the present invention, simple as ELISA method, but can high quick carry out nucleic acid
Molecular Detection, usefulness include, but are not limited to：First without Pretreated, sample is applied directly in plate hole, this
Not only simplify step, also avoid experimental error；Secondly, specific apparatus is not needed, each routine diagnosis laboratory can carry out；
3rd testing cost is relatively low.
More than, it is only the preferred embodiments of the invention, it is intended that further illustrate the present invention rather than it is defined.It is all
According to the simple replacement that above-mentioned word and attached drawing disclosure of that carry out, all claims in this patent
Within.
Claims (10)
1. a kind of multi-biotin signal amplification method, which is characterized in that by the connection of a multi-biotin molecule to letter to be detected
It number is amplified to promote the sensitivity of detection of nucleic acids, which includes the list by hybridizing identification attachment molecule
Ssdna molecule and the carrier molecule for biotin labeling.
2. multi-biotin signal amplification method as described in claim 1, which is characterized in that the carrier molecule is DNA, RNA, widow
Nucleotide, polynucleotides or polymer.
3. multi-biotin signal amplification method as claimed in claim 2, which is characterized in that the carrier molecule is single-stranded or double
Chain.
4. multi-biotin signal amplification method as claimed in claim 3, which is characterized in that the biotin on the carrier molecule is
Pass through enzyme reaction or the biotin of chemical reaction hybridization mark.
5. multi-biotin signal amplification method as claimed in claim 4, which is characterized in that the biotin labeling makes at least two
Biotin is covalently or non-covalently connected to the carrier.
6. multi-biotin signal amplification method as described in claim 1, which is characterized in that the attachment molecule is DNA, RNA
Or oligonucleotides.
7. multi-biotin signal amplification method as claimed in claim 6, which is characterized in that the attachment molecule is to hybridize with target
One or more synthetic molecules a part.
8. multi-biotin signal amplification method as claimed in claim 7, which is characterized in that the target molecule is to capture microwell plate
In upper or other solid phases.
9. multi-biotin signal amplification method as claimed in claim 8, which is characterized in that the target molecule is HBV-DNA.
10. multi-biotin signal amplification method as claimed in claim 8, which is characterized in that the target molecule is HCV-RNA.
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| CN201611086876.8A CN108118083A (en) | 2016-11-28 | 2016-11-28 | Multi-biotin signal amplification method |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111334611A (en) * | 2020-03-24 | 2020-06-26 | 武汉中帜生物科技股份有限公司 | Kit for detecting novel coronavirus (2019-nCoV) based on double amplification technology and application thereof |
-
2016
- 2016-11-28 CN CN201611086876.8A patent/CN108118083A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111334611A (en) * | 2020-03-24 | 2020-06-26 | 武汉中帜生物科技股份有限公司 | Kit for detecting novel coronavirus (2019-nCoV) based on double amplification technology and application thereof |
| CN111334611B (en) * | 2020-03-24 | 2021-09-24 | 武汉中帜生物科技股份有限公司 | Kit for detecting novel coronavirus (2019-nCoV) based on double amplification technology and application thereof |
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