CN102485906A - Dual-amplification method of template linear amplification and multi-biotin signal amplification - Google Patents
Dual-amplification method of template linear amplification and multi-biotin signal amplification Download PDFInfo
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- CN102485906A CN102485906A CN201010572588XA CN201010572588A CN102485906A CN 102485906 A CN102485906 A CN 102485906A CN 201010572588X A CN201010572588X A CN 201010572588XA CN 201010572588 A CN201010572588 A CN 201010572588A CN 102485906 A CN102485906 A CN 102485906A
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Abstract
The invention relates to a dual-amplification method of template linear amplification and multi-biotin signal amplification. The method comprises steps that: nucleic acid is separated from a specimen; T7RNA polymerase linear amplification is carried out, such that an RNA product is obtained; the RNA product obtained through linear amplification is captured onto a microtiter plate, and signal amplification is carried out. The method is advantaged in low cost and more reliable data. With the method, the sensitivity and the detection time are similar to those of a PCR method.
Description
Technical field
The present invention relates to the detection of living matter, relate in particular to detection the vital signs material.
Background technology
On the one hand, respiratory tract disease is caused by RNA (Yeast Nucleic Acid) virus, DNA (thymus nucleic acid) virus or bacterium.Confirm that the pathogenic agent kind is to selecting the specific aim medicine most important.Because the pathogen nucleic acid amount is few in the sample, need with template amplification such as PCR (polymerase chain reaction) or signal amplifies as bDNA technological (a chain nucleic acid signal amplifying technique) detects.
PCR is most commonly used to method of diagnosing.But, because some defectives in the PCR application process, expensive when detecting like non-linear amplification, false positive and PCR in real time, it is not the optimal mode of using.
The diagnosis of respiratory tract disease optionally reaches through template amplification, but behind the template amplification, needs hybridization detections of spending the night usually, like this for clinical be not a very suitable method just.
On the other hand; The disclosed a kind of multi-biotin signal amplification method of Chinese patent CN101792800A; Treat detection signal through the connection of a multi-biotin molecule and amplify to promote the sensitivity of detection of nucleic acids, this multi-biotin molecule comprises through the single stranded nucleic acid molecule of hybridization identification connector molecule and is used for biotin labeled carrier molecule.Thereby target molecule can be connected with link molecule, carrier molecule and a plurality of vitamin Hs and corresponding affinity tag and make the signal of detection obtain amplification.Connect a plurality of connector molecules in the different zones of target molecule, can obtain bigger amplification effect.
Summary of the invention
The technical problem that the present invention will solve be to overcome the deficiency of above-mentioned prior art and propose that a kind of cost is low, data are more reliable and susceptibility and detection time and the similar amplification method of PCR method.
The technical scheme that the present invention solves the problems of the technologies described above employing is, manufactures and designs the dual amplification method of a kind of template linear amplification and multi-biotin signal, and it comprises that step has: separate nucleic acid, isolating nucleic acid from sample; T7 RNA polymerase linear amplification obtains the RNA product; And, the RNA product of linear amplification is captured on the microwell plate, carry out signal and amplify.
In the present invention, be the situation of DNA for this nucleic acid, before linear amplification, also comprise: before adding transcriptase, adopt heating or NaOH/HCl to carry out sex change; Behind article one primer annealing, archaeal dna polymerase can be used for the dsDNA (double-stranded DNA) that synthetic one of first chain and second chain has a setting promotor.
In the present invention, be the situation of RNA for this nucleic acid, before linear amplification, also comprise the process that this RNA is converted into dsDNA.
The process that this RNA is converted into dsDNA comprises: the primer to have a setting promotor is transformed into cDNA (complementary DNA with ThermoScript II AMV (becoming the fiber viral reverse transcriptase from fowl) or MMLV (from the murine leukemia reversed transcriptive enzyme) with this RNA; Complementary DNA); Use RNase H (RNA enzyme H) to handle again; Make RNA/DNA become the cDNA of strand, under archaeal dna polymerase and the effect of second primer, with synthetic this dsDNA.
The process that this RNA is converted into dsDNA comprises: the primer to have a setting promotor is transformed into cDNA with ThermoScript II AMV or MMLV with this RNA; This cDNA is through 92 degree heat denatured, behind the second primer annealing, with this dsDNA of archaeal dna polymerase preparation.
The process that this RNA is converted into dsDNA comprises: the primer to have a setting promotor is transformed into cDNA with ThermoScript II AMV or MMLV with this RNA; This cDNA is through 92 degree heat denatured, and behind the second primer annealing, a circulation through PCR is with synthetic this dsDNA.
The process that this RNA is converted into dsDNA comprises: prepare dsDNA through a setting promotor and adopt NASBA (nucleic acid sequence based amplification; Amplification technique based on nucleotide sequence) step of describing; It comprises that specifically AMV ThermoScript II, RNase H and T7 RNA polymerase add with two primers, and wherein a primer has the T7 promotor.
This setting promotor is T7, T3 or SP6 promotor, perhaps T7, T3 or SP6 RNA polysaccharase.
In the present invention; The process that this signal amplifies specifically comprises: the connection through a multi-biotin molecule is amplified this RNA product, and this multi-biotin molecule comprises through the single strand dna of hybridization identification connector molecule and is used for biotin labeled carrier molecule.
Vitamin H on this carrier molecule is the vitamin H through enzyme reaction or chemical reaction hybridization mark.
Compare with prior art, the dual amplification method of template linear amplification of the present invention and multi-biotin signal, cost is low, data are more reliable and susceptibility and detection time and PCR method are similar.
Embodiment
Do further to detail below in conjunction with the most preferred embodiment shown in each accompanying drawing.
The dual amplification method of template linear amplification of the present invention and multi-biotin signal is that a kind of combination T7 amplification and multi-biotin signal amplify two amplification methods of (MBSA).Wherein, target nucleic acid is earlier by the T7 linear amplification, and amplified production is analyzed through MBSA.Different with PCR, T7 RNA linear amplification does not change nucleic acid-templated ratio, and the end product of its amplification is the RNA of strand, and RNA uses the MBSA detection by quantitative again.MBSA is the detection method of a kind of Gao Min, spends the night through hybridization, and it can detect 100 target molecules.After but target increased through t7 rna polymerase, hybridization just can be shortened in 2 hours.
This dual amplification method, the step that comprises has:
1, separate nucleic acid
Isolating nucleic acid from sample.No matter RNA or DNA, with commercial separate nucleic acid test kit, like the nucleic acid extracting with NucliSens Extractor (full-automatic nucleic acid extraction appearance) (BioMerieux, Boxtel, The Netherland).
2, T7 amplification
If template is RNA, there are many methods to can be used to transform, before the T7RNA amplification, make it to become dsDNA.
A, with ThermoScript II AMV or MMLV RNA is transformed into cDNA with the primer that has the T7 promotor, handles with RNase H, make RNA/DNA become the cDNA of strand, under archaeal dna polymerase and the effect of second primer, it can be as the template of synthesizing dsDNA.
B, cDNA behind the second primer annealing, prepare dsDNA with archaeal dna polymerase through the 92C heat denatured, also can reach through the circulation of PCR.
C, the third method, preparing dsDNA through the T7 promotor is the method steps of describing with NASBA.In this method, AMV ThermoScript II, RNase H and T7 RNA polymerase add with two primers, and wherein a primer has the T7 promotor.
If target is DNA, before adding transcriptase, need sex change, sex change can be through heating or NaOH/HCl; Behind article one primer annealing, archaeal dna polymerase can be used for the synthetic of first chain and second chain.
The dsDNA that has the T7 promotor just can be transcribed into RNA under the effect of T7 RNA polymerase.
Above-mentioned T7 promotor or T7 RNA polysaccharase can be used other promotor such as T3 and SP6 and T3 and SP6 RNA polysaccharase.
3, signal amplifies
The RNA product of amplification captures on the microwell plate, amplifies or other method for amplifying signal detection with the multi-biotin signal.
The process that this signal amplifies specifically comprises: the connection through a multi-biotin molecule is amplified this RNA product, and this multi-biotin molecule comprises through the single strand dna of hybridization identification connector molecule and is used for biotin labeled carrier molecule.
Vitamin H on this carrier molecule is the vitamin H through enzyme reaction or chemical reaction hybridization mark.
The dual amplification method of template linear amplification of the present invention and multi-biotin signal is compared with PCR in real time, and have many advantages: cost is low, and this method does not need expensive instrument and expensive fluorescent probe; This method can not change the ratio of template in amplification, thereby data are more reliable; Susceptibility and detection time and PCR are similar.
More than, be merely the present invention's preferred embodiment, be intended to further specify the present invention, but not it is limited.All simple substitution of carrying out according to above-mentioned literal and the disclosed content of accompanying drawing are all within the claim protection domain of this patent.
Claims (10)
1. the dual amplification method of template linear amplification and multi-biotin signal is characterized in that, comprises that step has:
Separate nucleic acid, isolating nucleic acid from sample;
T7 RNA polymerase linear amplification obtains the RNA product; And,
The RNA product of linear amplification is captured on the microwell plate, carry out signal and amplify.
2. dual amplification method as claimed in claim 1 is characterized in that, is the situation of DNA for this nucleic acid, before linear amplification, also comprises: before adding transcriptase, adopt heating or NaOH/HCl to carry out sex change; Behind article one primer annealing, archaeal dna polymerase can be used for the dsDNA that synthetic one of first chain and second chain has a setting promotor.
3. dual amplification method as claimed in claim 1 is characterized in that, is the situation of RNA for this nucleic acid, before linear amplification, also comprises the process that this RNA is converted into dsDNA.
4. dual amplification method as claimed in claim 3; It is characterized in that; The process that this RNA is converted into dsDNA comprises: the primer to have a setting promotor is transformed into cDNA with ThermoScript II AMV or MMLV with this RNA, handles with RNase H again, makes RNA/DNA become the cDNA of strand; Under archaeal dna polymerase and the effect of second primer, with synthetic this dsDNA.
5. dual amplification method as claimed in claim 3 is characterized in that, the process that this RNA is converted into dsDNA comprises: the primer to have a setting promotor is transformed into cDNA with ThermoScript II AMV or MMLV with this RNA; This cDNA is through 92 degree heat denatured, behind the second primer annealing, with this dsDNA of archaeal dna polymerase preparation.
6. dual amplification method as claimed in claim 3 is characterized in that, the process that this RNA is converted into dsDNA comprises: the primer to have a setting promotor is transformed into cDNA with ThermoScript II AMV or MMLV with this RNA; This cDNA is through 92 degree heat denatured, and behind the second primer annealing, a circulation through PCR is with synthetic this dsDNA.
7. dual amplification method as claimed in claim 3; It is characterized in that; The process that this RNA is converted into dsDNA comprises: set promotor through one and prepare the step that dsDNA is employing NASBA description; It comprises that specifically AMV ThermoScript II, RNase H and T7 RNA polymerase add with two primers, and wherein a primer has the T7 promotor.
8. like claim 2,4,5,6 or 7 described dual amplification methods, it is characterized in that this setting promotor is T7, T3 or SP6 promotor, perhaps T7, T3 or SP6 RNA polysaccharase.
9. dual amplification method as claimed in claim 1; It is characterized in that; The process that this signal amplifies specifically comprises: the connection through a multi-biotin molecule is amplified this RNA product, and this multi-biotin molecule comprises through the single strand dna of hybridization identification connector molecule and is used for biotin labeled carrier molecule.
10. dual amplification method as claimed in claim 9 is characterized in that, the vitamin H on this carrier molecule is the vitamin H through enzyme reaction or chemical reaction hybridization mark.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103361438A (en) * | 2013-07-30 | 2013-10-23 | 武汉中帜生物科技有限公司 | Method for detecting nucleic acid by combining template amplification and signal amplification |
| CN103525942A (en) * | 2013-10-31 | 2014-01-22 | 武汉中帜生物科技有限公司 | Nucleic acid detection method combining RNA amplification with hybrid capture method |
| CN110923362A (en) * | 2019-12-19 | 2020-03-27 | 武汉中帜生物科技股份有限公司 | Colloidal gold chromatography kit for simultaneously detecting herpes simplex virus I/II and application thereof |
| CN111304295A (en) * | 2019-12-19 | 2020-06-19 | 武汉中帜生物科技股份有限公司 | Kit for simultaneously detecting nucleic acids of neisseria gonorrhoeae, chlamydia trachomatis and ureaplasma urealyticum and application thereof |
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2010
- 2010-12-03 CN CN201010572588XA patent/CN102485906A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103361438A (en) * | 2013-07-30 | 2013-10-23 | 武汉中帜生物科技有限公司 | Method for detecting nucleic acid by combining template amplification and signal amplification |
| CN103361438B (en) * | 2013-07-30 | 2016-09-28 | 武汉中帜生物科技股份有限公司 | A kind of template amplification and signal amplify the method combining detection nucleic acid |
| CN103525942A (en) * | 2013-10-31 | 2014-01-22 | 武汉中帜生物科技有限公司 | Nucleic acid detection method combining RNA amplification with hybrid capture method |
| CN110923362A (en) * | 2019-12-19 | 2020-03-27 | 武汉中帜生物科技股份有限公司 | Colloidal gold chromatography kit for simultaneously detecting herpes simplex virus I/II and application thereof |
| CN111304295A (en) * | 2019-12-19 | 2020-06-19 | 武汉中帜生物科技股份有限公司 | Kit for simultaneously detecting nucleic acids of neisseria gonorrhoeae, chlamydia trachomatis and ureaplasma urealyticum and application thereof |
| CN111304295B (en) * | 2019-12-19 | 2022-08-05 | 武汉中帜生物科技股份有限公司 | Kit for simultaneously detecting nucleic acids of neisseria gonorrhoeae, chlamydia trachomatis and ureaplasma urealyticum and application thereof |
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Application publication date: 20120606 |