OA20437A - Nucleic acid release agent, nucleic acid PCR amplification method and PCR amplification kit - Google Patents
Nucleic acid release agent, nucleic acid PCR amplification method and PCR amplification kit Download PDFInfo
- Publication number
- OA20437A OA20437A OA1202100324 OA20437A OA 20437 A OA20437 A OA 20437A OA 1202100324 OA1202100324 OA 1202100324 OA 20437 A OA20437 A OA 20437A
- Authority
- OA
- OAPI
- Prior art keywords
- sec
- sample
- nucleic acid
- pcr amplification
- amplification
- Prior art date
Links
- 230000003321 amplification Effects 0.000 title claims description 103
- 238000003199 nucleic acid amplification method Methods 0.000 title claims description 103
- 150000007523 nucleic acids Chemical class 0.000 title claims description 95
- 108020004707 nucleic acids Proteins 0.000 title claims description 94
- 239000003795 chemical substances by application Substances 0.000 title claims description 62
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 32
- FAPWRFPIFSIZLT-UHFFFAOYSA-M sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 32
- KWIUHFFTVRNATP-UHFFFAOYSA-N Trimethylglycine Chemical compound C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 claims description 26
- 238000006243 chemical reaction Methods 0.000 claims description 24
- 229920004890 Triton X-100 Polymers 0.000 claims description 21
- -1 comprising Tris-HCI Substances 0.000 claims description 19
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 17
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 17
- 241000700605 Viruses Species 0.000 claims description 16
- 239000001103 potassium chloride Substances 0.000 claims description 16
- 235000011164 potassium chloride Nutrition 0.000 claims description 16
- 239000011780 sodium chloride Substances 0.000 claims description 15
- 229920001223 polyethylene glycol Polymers 0.000 claims description 14
- 235000002639 sodium chloride Nutrition 0.000 claims description 14
- 102100001249 ALB Human genes 0.000 claims description 13
- 101710027066 ALB Proteins 0.000 claims description 13
- 241000283690 Bos taurus Species 0.000 claims description 13
- 239000002202 Polyethylene glycol Substances 0.000 claims description 13
- 229940050528 albumin Drugs 0.000 claims description 13
- 229960003237 betaine Drugs 0.000 claims description 13
- 238000010839 reverse transcription Methods 0.000 claims description 13
- 238000001816 cooling Methods 0.000 claims description 12
- 238000002156 mixing Methods 0.000 claims description 12
- 230000000241 respiratory Effects 0.000 claims description 12
- YFVGRULMIQXYNE-UHFFFAOYSA-M lithium;dodecyl sulfate Chemical compound [Li+].CCCCCCCCCCCCOS([O-])(=O)=O YFVGRULMIQXYNE-UHFFFAOYSA-M 0.000 claims description 11
- 241000711549 Hepacivirus C Species 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 8
- 241000894006 Bacteria Species 0.000 claims description 7
- 102000004190 Enzymes Human genes 0.000 claims description 7
- 108090000790 Enzymes Proteins 0.000 claims description 7
- 230000000968 intestinal Effects 0.000 claims description 7
- 238000005119 centrifugation Methods 0.000 claims description 6
- 239000004698 Polyethylene (PE) Substances 0.000 claims description 4
- 230000004913 activation Effects 0.000 claims description 4
- 230000036425 denaturation Effects 0.000 claims description 4
- 238000004925 denaturation Methods 0.000 claims description 4
- 238000002844 melting Methods 0.000 claims description 4
- 229920000573 polyethylene Polymers 0.000 claims description 4
- 238000006911 enzymatic reaction Methods 0.000 claims description 3
- 239000002244 precipitate Substances 0.000 claims description 3
- 229920000160 (ribonucleotides)n+m Polymers 0.000 description 33
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Chemical group [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 27
- 239000000243 solution Substances 0.000 description 24
- 238000003753 real-time PCR Methods 0.000 description 18
- 230000000052 comparative effect Effects 0.000 description 14
- 230000035945 sensitivity Effects 0.000 description 12
- 210000004027 cells Anatomy 0.000 description 10
- 238000009835 boiling Methods 0.000 description 8
- 230000009089 cytolysis Effects 0.000 description 8
- 230000002934 lysing Effects 0.000 description 8
- 230000002633 protecting Effects 0.000 description 8
- 108010092799 EC 2.7.7.49 Proteins 0.000 description 7
- 102000033147 ERVK-25 Human genes 0.000 description 7
- 239000011324 bead Substances 0.000 description 7
- 238000000605 extraction Methods 0.000 description 7
- 238000000746 purification Methods 0.000 description 7
- 229920003013 deoxyribonucleic acid Polymers 0.000 description 4
- 238000010438 heat treatment Methods 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 210000003800 Pharynx Anatomy 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 238000011109 contamination Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000007850 fluorescent dye Substances 0.000 description 3
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 3
- 239000011591 potassium Substances 0.000 description 3
- 229910052700 potassium Inorganic materials 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- 230000000593 degrading Effects 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 241001493065 dsRNA viruses Species 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000003834 intracellular Effects 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 239000012139 lysis buffer Substances 0.000 description 2
- 239000006249 magnetic particle Substances 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- SBASXUCJHJRPEV-UHFFFAOYSA-N 2-(2-Methoxyethoxy)ethanol Chemical compound COCCOCCO SBASXUCJHJRPEV-UHFFFAOYSA-N 0.000 description 1
- 210000004369 Blood Anatomy 0.000 description 1
- 210000000349 Chromosomes Anatomy 0.000 description 1
- 241000450599 DNA viruses Species 0.000 description 1
- 101700011961 DPOM Proteins 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N HCl Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- 101710029649 MDV043 Proteins 0.000 description 1
- 239000008118 PEG 6000 Substances 0.000 description 1
- 101700061424 POLB Proteins 0.000 description 1
- 210000002381 Plasma Anatomy 0.000 description 1
- 229920002584 Polyethylene Glycol 6000 Polymers 0.000 description 1
- 206010036790 Productive cough Diseases 0.000 description 1
- 101700054624 RF1 Proteins 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 108060006413 Shal Proteins 0.000 description 1
- 235000015076 Shorea robusta Nutrition 0.000 description 1
- 240000007944 Shorea robusta Species 0.000 description 1
- 210000003802 Sputum Anatomy 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Tris Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- UIIMBOGNXHQVGW-UHFFFAOYSA-M buffer Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 239000005549 deoxyribonucleoside Substances 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 229910000041 hydrogen chloride Inorganic materials 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- KEAYESYHFKHZAL-UHFFFAOYSA-N sodium Chemical compound [Na] KEAYESYHFKHZAL-UHFFFAOYSA-N 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 235000011178 triphosphate Nutrition 0.000 description 1
- 239000001226 triphosphate Substances 0.000 description 1
- UNXRWKVEANCORM-UHFFFAOYSA-I triphosphate(5-) Chemical compound [O-]P([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O UNXRWKVEANCORM-UHFFFAOYSA-I 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Description
NUCLEIC ACID RELEASE AGENT, NUCLEIC ACID PCR AMPLIFICATION METHOD AND PCR AMPLIFICATION KIT
TECHNICAL FIELD
The présent disclosure relates to the field of moiecular biology, in particular to a nucleic acid release agent, a method for PCR amplification of a nucleic acid and a PCR amplification kit
BACKGROUND
Polymerase chain réaction (PCR) is a molecular biological technique for amplifying a spécifie nucleic acid fragment, împortantîy chafacterized by massive enrichment and increase of a trace amount of nucleic acid, so as to facilitate the détection of the trace amount of nucleic acid. The commonly used PCR method in medical diagnosis is mainly a real-time fluorescent quantitative PCR (qPCR) method based on dual fluorescent probes. Targets for in vitro diagnosis using the qPCR method mainly include human genomic DNA, DNA viruses, bacteria, fungt RNA viruses, and the like. However, since RNA has a singlestranded structure, which is unstable and easily degraded, a process of sample treatment is subjected to very stringent requirements, and a more complicated method is required for pretreatment, nucleic acid extraction and purification of the RNA sample to be amplified to obtain the pure nucleic acid, then the détection can be performed to obtain a stable resuit.
SUMMARY
Accordingly, it is necessary to provide a nucleic acid release agent that can simplify the PCR amplification method of RNA samples.
The présent disclosure provides a nucleic acid release agent comprising Tris-HCI, sodium chloride, potassium chîoride, Tween 20, Triton X-100, ethy! phenyl polyethylene glycol and a strong base; wherein Tris-HCIhas a molar concentration ranging from 0.5 mM to 500 mM, sodium chloride has a molarconcentration from 20 mM to 500 mM, potassium chloride has a mass concentration ranging from 5 mg/mL to 8 mg/mL, Tween 20 has a volume percentage ranging from 0.1% to 2%, Triton X-100 has a volume percentage ranging from 0.1% to 3%, ethyl phenyl polyethylene glycol has a volume percentage ranging from 0.1 % to 3%, and the strong base has a mass concentration ranging from 2 mg/mL to 50 mg/mL. ïn the nucleic acid release agent ofthe présent disciosure, a certain proportion ofthe strong base lyses cells, which release nucleic acids. Sodium chloride and potassium chloride protect nucleic acids by coordinating the balance of intracellular and extracelluîar ions. Tris
HCl is for keeping a pH value stable during cell lysis and better compatible with a PCR reaction solution in subséquent amplification. Triton X-100, on the one hand, can protect nucleic acids, especiaiiy singie-stranded RNA, aiiowing RNA to be stored in an alkaline environment. On the other hand, Triton X-100 with potassium chloride in a certain ratio can reduce the inhibitory effect of the strong alkaline environment on the enzyme in the PCR reaction, thereby ensuring the amplification efficiency of RNA. Tween 20 and ethyl phenyl polyethylene glycol can protect reverse transcriptase, so that the reverse transcriptase works normally in the alkaline environment. Through the synergy of the above components, the nucleic acid release agent of the présent disclosure enables a sample containing RNA to directly release RNA at room température, thereby avoiding the problem of sample contamination caused by aérosol generated by heating, and effectively preventing RNA from degrading in the alkaline environment. More importantly, it can be directiy mixed with the PCR reaction solution for PCR amplification to complété the amplification without the need for compiicated nucleic acid extraction and purification processes. This truly realizes a one-chamber, pollution-free, simple and rapid RNA amplification and détection, with high détection sensitivity and good repeatability.
In one of the embodiments, the nucleic acid release agent further comprises betaine and bovine sérum albumin, wherein betaine has a mass concentration ranging from 0.1 mg/mL to 20 mg/mL, and bovine sérum albumin has a mass concentration ranging from 5 mg/mL to 100 mg/mL.
In one of the embodiments, the nucleic acid release agent further comprises protéinase K and lithium dodecyl sulfate, wherein protéinase K has a mass concentration ranging from 0.02 mg/mL to 1.5 mg/mL, and lithium dodecyl sulfate has a mass concentration ranging from 0.4 mg/mL to 30 mg/mL.
The présent disclosure also provides a method for PCR amplification of a nucleic acid, comprising steps of: mixing the nucleic acid release agent as described above with a sample, placing the mixture at 25°C to 60°C for 2 min to 10 min, and adding a PCR reaction solution for PCR amplification.
In one of the embodiments, the method further comprises a sample pretreatment step of mixing the sample with polyethylene glycol followed by centrifugation to collect a precipitate before mixing the nucleic acid release agent with the sample.
In one of the embodiments, the PCR amplification of intestinal viruses in the sample is performed at following conditions:
reverse transcription at 48°C to 52°C for 28 min to 32 min;
thermal dénaturation at 93°C to 97°C for 0.9 min to 1.1 min;
several cycles of amplification at 93°C to 97°C for 13 sec to 17 sec followed by
53°C to 57°C for 28 sec to 32 sec;
cooling at 23°C to 27°C for 8 secto 12 sec.
in one of the embodiments, the PCR amplification of hepatitis C virus in the sample is performed at following conditions:
pre-denaturation and enzyme activation at 93 °C to 97°C for 0.9 min to 1.1 min;
reverse transcription at 58°C to 62°C for 28 min to 32 min;
thermal dénaturation at 93°C to 97°C for 0.9 min to 1,1 min;
several cycles of amplification at 93°C to 97°C for 13 sec to 17 sec followed by
58°C to 62°C for 28 sec to 32 sec;
cooling at 23°C to 27°C for 8 sec to 12 sec.
In one of the embodiments, the PCR amplification of respiratory viruses in the sample is performed at following Conditions:
reverse transcription at 48°C to 52°C for 28 min to 32 min;
thermal dénaturation at 93°C to 97°C for 0.9 min to 1.1 min;
several cycles of amplification at 93°C to 97°C for 13 sec to 17 sec followed by
58°C to 62°C for 28 sec to 32 sec;
cooling at 23°C to 27°C for 8 sec to 12 sec.
in one of the embodiments, the PCR amplification of respiratory bacteria in the sample is performed at following conditions:
UDG enzyme réaction at 48°C to 52°C for 1.9 min to 2.1 min;
thermal dénaturation at 92°C to 96°C for 2.9 min to 3.1 min;
several cycles of amplification at 92°C to 96°C for 8 sec to 12 sec followed by
58°C to 62QC for 18 sec to 22 sec;
extension and fluorescence collection at 73°C to 77°C for 18 sec to 22 sec;
melting curve: 62°C to 75°C.
The present disclosure also provides a PCR amplification kit, comprising the nucleic acid release agent as described above and a PCR reaction solution.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows real-time fluorescence quantitative PCR amplification curves in Examples 125.
FIG. 2 shows real-time fluorescence quantitative PCR amplification curves in Examples 26-50.
FIG. 3 shows real-time fluorescence quantitative PCR amplification curves in Examples 51-70.
FIG. 4 shows real-time fluorescence quantitative PCR amplification curves in Examples 71-90.
DETAILED DESCRIPTION OF THE EMBODIMENTS
To make the présent disclosure easy to understand, a more comprehensive description of the présent disclosure will be given below, and better embodiments of the présent disclosure are given below. However, the présent disclosure can be implemented in many different forms and shal! not be limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to provide a more thorough and comprehensive understanding of the disclosure of the présent disclosure.
Unless otherwise defined, ail technical and scientific terms used herein hâve the same meanings as those generally understood by those skilled in the art of the présent disclosure. The terms used in the spécification of the présent disclosure are only for the purpose of describing spécifie embodiments, rather than limiting the présent disclosure. The term “and/or” as used herein includes any and ail combinations of one or more reïated listed items.
In an embodiment of the présent disclosure, a nucleic acid release agent comprises TrisHCI, sodium chloride, potassium chloride, Tween 20, Triton X-100, ethyl phenyl poiyethylene glycol and a strong base; wherein Tris-HCI has a moiar concentration ranging from 0.5 mM to 500 mM, sodium chloride has a moiar concentration from 20 mM to 500 mM, potassium chloride has a mass concentration ranging from 5 mg/mL to 8 mg/mL, Tween 20 has a volume percentage ranging from 0.1% to 2%, Triton X-100 has a volume percentage ranging from 0.1% to 3%, ethyl phenyl poiyethylene glycol has a volume percentage ranging from 0.1% to 3%, and the strong base has a mass concentration ranging from 2 mg/mL to 50 mg/mL.
In order to achieve the effect of lysis, a lysis solution needs to be alkaline, but RNA is easily degraded in an alkaline environment. Moreover, a reverse transcriptase, which is required for PCR amplification and détection of RNA, is relatively fragile compared with the thermally stable TaqDNA polymerase and is easily inhibited in an alkaline environment, resulting in an inability to achieve the effect of reverse transcription. Therefore, RNA in the lysis solution generally cannot be directly amplified and detected by PCR. At présent, pre-treating methods for PCR amplification and détection of RNA samples mainly include a boiling lysis method and a magnetic bead method. in the boiling lysis method, nucleic acids in a sample are released by boiling under the action of a lysis buffer and dissolved in the lysis buffer. While the cells are lysed in a boiling water bath, the proteins and chromosomes of the cells are denatured. Then the denatured proteins and other impurities are removed by centrifugation, and the nucleic acids in the supematant are recovered for PCR amplification. However, coagulation of the proteins subjected to boiling at a high température causes part of the nucleic acids to be wrapped and run off with the centrifugation, leading directly to a decrease in the amount of template nucleic acids in the supematant, which reduces the sensitivity of subséquent amplification and détection. In addition, aérosols are easily produced by boiling and heating, leading to sample contamination, which will resuit in falsepositive results in subséquent détection. In the magnetic bead method, cells are lysed in a lysis solution, and free nucleic acid molécules are specificaîly adsorbed to the surface of the magnetic particles, while impurities such as proteins are not adsorbed but remained in the solution. After reacting for a certain period of time, the magnetic particles are separated from the liquid under the action of a magnetic field, followed by elution with an eluent to obtain pure nucleic acids. However, the magnetic bead method has complicated operation, requires a sample size that is generally 200 to 600 microliters, and has a very high demand on equipments, giving limitations on its promotion and use.
Based on the above mechanism, in the nucleic acid release agent of this embodiment, a certain proportion of the strong base lyses cells, which release nucleic acids.. Sodium chloride and potassium chloride protect nucleic acids by coordinating the balance of intracellular and extracellular ions. Tris-HCI is for keeping a pH value stable during cell lysis and is better compatible with a PCR reaction solution in subséquent amplification. Triton X-100, on the one hand, can protect nucleic acids, especially single-stranded RNA, allowing RNAto be stored in an alkaline environment. On the other hand, Triton X-100 with potassium chloride in a certain ratio can reduce the inhibitory effect of the strong alkaline environment on the enzyme in the PCR reaction, thereby ensuring the amplification efficiency of RNA. Tween 20 and ethyl phenyl polyethylene glycol can protect reverse transcriptase, so that the reverse transcriptase works normally in the alkaline environment. Through the synergy of the above components, the nucleic acid release agent of this embodiment enables a sample containing RNA to directly release RNA from at room température, thereby avoiding the problem of sample contamination caused by aérosol generated by heating and effectively preventing RNA from degrading in the alkaline environment. More importantly, it can be directly mixed with the PCR reaction solution for PCR amplification to complète the amplification, without the need for complicated nucleic acid extraction and purification processes. This truly realizes a one-chamber, pollution-free, simple and rapid RNA amplification and détection, with high détection sensitivity and good repeatability. It can be understood that the nucleic acid release agent can be used not only for PCR amplification and détection of RNA samples, but also for multiple and combined amplification and détection of DNA samples or mixed samples of RNA and DNA.
Specificaîly, the sirong base is sodium hydroxide or potassium hydroxide, and the like, which can be selected as required.
In a spécifie example, the nucleic acid release agent further comprises betaine and bovine sérum albumin, wherein betaine has a mass concentration ranging from 0.1 mg/mL to 20 mg/mL, and bovine sérum albumin has a mass concentration ranging from 5 mg/mL to 100 mg/mL. The certain proportion of betaine and bovine sérum albumin added may collaborate with Triton X-100 to better protect RNA under aîkaline conditions and prevent polymerase and reverse transcriptase from their dénaturation, thereby guaranteeing rapid release and amplification of RNA to achieve rapid détection of RNA samples.
In a spécifie example, the nucleic acid release agent further comprises protéinase K and lithium dodecyl sulfate, wherein protéinase K has a mass concentration ranging from 0.02 mg/mL to 1.5 mg/mL, and lithium dodecyl sulfate has a mass concentration ranging from 0.4 mg/mL to 30 mg/mL. The certain proportion of protéinase K and lithium dodecyl sulfate added may dénature and dégradé RNase, thereby further protecting RNA from its dégradation.
In an embodiment of the présent disclosure, a method for PCR amplification of a nucleic acid comprises steps of: mixing the nucleic acid release agent as described above with a sample, placing the mixture at25°Cto 60°C for 2 min to 10 min, and adding a PCR reaction solution for PCR amplification.
The method for PCR amplification of the nucleic acid in this embodiment realizes the operation of direct amplification without extraction and purification for nucleic acid samples such as RNA, that is, the amplification and détection of the nucleic acid sample can be done by adding the nucleic add release agent described above to the sample to release the nucleic acids from the cells, adding the PCR reaction solution and performing PCR amplification such as real-time fluorescent quantitative PCR directly, without the need for boiling and heating or extraction and purification processes, and the like. This truly realizes a one-chamber, pollution-free, simple and rapid RNA amplification and détection, with high détection sensitivity and good repeatability.
In a spécifie example, a volume ratio ofthe nucleic acid release agent to the sample is 1:1 to 1:5. Optionaliy, the samples can be of a variety of types inciuding sérum, plasma, an oropharyngeal swab, a nasopharyngeal swab, alveolar lavage fluid, stool and the like, and after being mixed with the nucleic acid release agent, can be directly used in détection methods downstream, such as PCR amplification or gene chip.
In a spécifie example, the PCR reaction solution comprises deoxyribonucleoside triphosphate, a forward primer, a reverse primer, DNA polymerase, reverse transcriptase and amplification buffer, and the iike. it can be understood that, according to different types and purposes of the PCR reaction, the composition of the PCR reaction solution can be selected as needed and is not limited thereto. For example, when performing reai-time fluorescent quantitative PCR, the PCR reaction solution also includes fluorescent probes or fluorescent dyes, etc.
In a spécifie exampie, the method further comprises a sample pretreatment step of mixing the sample with poiyethyiene glycoi followed by centrifugation to colled a precipitate before mixing the nucleic acid release agent with the sample. Specificaily, poiyethyiene glycoi is PEG-6000, with a concentration of 0.5% to 5% by volume, Poiyethyiene glycoi as a nucleic acid sédimentation agent can be used for the treatment of compiex samples. It can effectively capture RNA viruses in free form and increase the sensitivity of later détection without affecting the PCR reaction system, and can significantly improve the performance of rapid PCR détection of RNA. For example, for cell preserving fiuids containing high-salt solutions, virus preserving fiuids, or hemolyzed blood samples, 50 to 5000 microliters of the sample can be taken and PEG solution in an equal volume can be added thereto followed by centrifugation at 3000 to 13000 rpm/min for 1 to 10 min, with the supematant discarded and the pellet left, and then 50 to 100 microliters of nucleic acid release agent is added.
In a spécifie example, the PCR amplification of intestinal viruses in the sample is performed at following conditions:
reverse transcription at 48QC to 52°C for 28 min to 32 min;
thermal dénaturation at 93°C to 97°C for 0.9 min to 1.1 min;
several cycles of amplification at 93°C to 97°C for 13 sec to 17 sec followed by 53°C to 57°C for 28 sec to 32 sec;
cooling at 23°C to 27°C for 8 sec to 12 sec,
In a spécifie example, the PCR amplification of hepatitis C virus (HCV) in the sample is performed at following conditions:
pre-denaturation and enzyme activation at 93°C to 97°C for 0.9 min to 1.1 min; reverse transcription at 58°C to 62°C for 28 min to 32 min;
thermal dénaturation ai 93OC to 97°C for 0.9 min to 1.1 min;
several cycles of amplification at 93°C to 97°C for 13 sec to 17 sec followed by 58°C to 62°C for 28 sec to 32 sec;
cooling at 23°C to 27°C for 8 sec to 12 sec.
In a spécifie example, the PCR amplification of respiratory viruses in the sample is performed at following conditions:
reverse transcription at 48°C to 52°C for 28 min to 32 min;
thermal dénaturation at 93°C to 97°C for 0.9 min to 1.1 min;
several cycles of amplification at 93°C to 97°C for 13 sec to 17 sec followed by
58°C to 62°C for 28 sec to 32 sec;
cooling at 23°C to 27°C for 8 sec to 12 sec.
In a spécifie example, the PCR amplification of respiratory bacteria in the sample is performed at following conditions:
UDG enzyme reaction at 48°C to 52°C for 1.9 min to 2.1 min;
thermal dénaturation at 92°C to 96°C for 2.9 min to 3.1 min;
several cycles of amplification at 92°C to 96 °C for 8 sec to 12 sec followed by
58°C to 62°C for 18 sec to 22 sec;
extension and fluorescence collection at 73°C to 77°C for 18 sec to 22 sec; melting curve: 62°C to 75°C.
It can be understood that the conditions for PCR amplification are not limited to the above spécifie examples, and can be adjusted according to different PCR types and purposes.
In an embodiment of the présent disclosure, a PCR amplification kit comprises the nucleic acid release agent as described above and a PCR reaction solution. The PCR amplification kit in this embodiment realizes the operation of direct amplification without extraction and purification for nucleic acid samples such as RNA, that is, the amplification and détection of the nucleic acid sample can be done by adding the nucleic acid release agent described above to the sample to release the nucleic acids from the cells, adding the PCR reaction solution and performing PCR amplification such as real-time fluorescent quantitative PCR directly, without the need for boiling and Keating or extraction and purification processes, and the like. This truly realizes a one-chamber, pollution-free, simple and rapid RNA amplification and détection, with high détection sensitivity and good repeatability.
The following are spécifie examples.
1. Détection for intestinal viruses
Examples 1 to 25: 25 intestinal virus throat swab samples (virus preserving fluid vehicle) were prepared. 100pL of each sample was taken and centrifugated at 12000rpm/min for 10min, with the supematani discarded. After adding 50 pL of a nucleic acid release agent and standing for 10 min, 10 pL of the nucleic acid release agent-treated sample was mixed with 40 pL of a PCR reaction solution for performing real-time fluorescence quantitative PCR amplification. The amplification curve is shown in FIG. 1. The nucleic acid release agent used in Examples 1 to 25 comprised Tris-HCI, sodium chloride, potassium chloride,
Tween 20, Triton X-100, ethyl phenyl polyethylene glycol, betaine, bovine sérum albumin, Protéinase K, lithium dodecyl sulfate and sodium hydroxide; wherein Tris-HCI had a molar concentration of 0.5 mM, sodium chioride had a molar concentration of 500 mM, Tween 20 had a volume percentage of 0.1%, Triton X-100 had a volume percentage of 3%, ethyl phenyl polyethylene glycol had a volume percentage of 0.1%, potassium chioride had a mass concentration of 8 mg/mL, sodium hydroxide had a mass concentration of 2 mg/mL, betaine had a mass concentration of 20 mg/mL. bovine sérum albumin had a mass concentration of 5 mg/mL, protéinase K had a mass concentration of 1.5 mg/mL, and lithium dodecyl sulfate had a mass concentration of 0.4 mg/mL.
Comparative Examples 1 -25: The above 25 samples were treated by the magnetic bead method, and real-time fluorescent quantitative PCR amplification was performed. Comparative Exampies 26-35 were basicaily the same as Exemples 1 to 10, except that Tween 20 and ethyl phenyl polyethylene glycol were not comprised in the nucleic acid release agent.
Comparative Examples 36-45 were basicaily the same as Examples 11 to 20, except that Triton X-100 was not comprised in the nucleic acid release agent. Comparative Examples 46-50 were basicaily the same as Examples 21 to 25, except that Triton X-100 in the nucleic acid release agent had a volume percentage of 8%, and potassium chioride had a mass concentration of 15 mg/mL
Ct values in the Exemples and Comparative Examples are shown in Table 1. PCR amplification was performed at following conditions: reverse transcription at 50°C for 30 min;
thermal dénaturation at 95°C for 1 min;
cycles of amplification at 95°C for 15 sec followed by 55°C for 30 sec; cooling at 25°C for 10 sec.
Table 1
| Comparative Examples 1 to 13 | Examples 1 to 13 | Comparative Examples 14 to 25 | Examples 14 to 25 | ||
| Sample 1 | 24.49 | 24.07 | Sample 14 | 20.06 | 21.68 |
| Sample 2 | 25.88 | 25.32 | Sample 15 | 24.21 | 20.64 |
| Sample 3 | 25.55 | 23.61 | Sample 16 | 23.30 | 23.63 |
| Sample 4 | 23.89 | 23.55 | Sample 17 | 26.39 | 27.63 |
| Sample 5 | 27.36 | 26.06 | Sample 18 | 29.66 | 28.03 |
| Sample 6 | 26.61 | 28.12 | Sample 19 | 24.24 | 21.44 |
| Sample 7 | 28.56 | 29.06 | Sample 20 | 34.01 | 34.23 |
| Sample 8 | 44.09 | 37.96 | Sample 21 | 28.69 | 27.57 |
| Sample 9 | 32.44 | 31.82 | Sample 22 | 27.10 | 25.31 |
| Sample 10 | 30.04 | 29.27 | Sample 23 | 29.63 | 31.11 |
| Sample 11 | 22.54 | 24.24 | Sample 24 | 31.99 | 32.50 |
| Sample 12 | 24.18 | 25.83 | Sample 25 | 27.69 | 27.60 |
| Sample 13 | 31.13 | 29.93 |
According to the test results, samples that were positive for intestinal virus can be detected in ail Comparative Examples 1 to 25 and Examples 1 to 25 with a consistent rate of results of 100% and good accuracy. However, the samples that were positive for intestinal virus cannot be stably and successfully detected in Comparative Examples 26-50. In addition, a smaller Ct value indicates higher détection sensitivity. Comparing the Ct values, it can be seen that in the examples using the nucleic acid release agent and the method for PCR amplification ofthe nucleic acid ofthe présent disclosure, the sensitivity ofthe amplification and détection of RNA samples is équivalent to that using the magnetic bead method.
2. Combined détection for respiratory bacteria
Exemples 26 to 50: 25 respiratory tract sputum samples (normal saline vehicle) were prepared. 5 pL of each sample was taken, and 5 pL of a nucleic acid release agent was added thereto, followed by standing for 10 min. 40 pL of a PCR reaction solution was added with mixing for performing real-time fluorescence quantitative PCR amplification. The amplification curve is shown in FIG, 2. The nucleic acid release agents used comprised Tris-HCI, sodium chloride, potassium chloride, Tween 20, Triton X-100, ethyl phenyl polyethylene glycof, betaine, bovine sérum albumin, protéinase K, lithium dodecyl sulfate and sodium hydroxide; wherein Tris-HCI had a molar concentration of 500 mM, sodium chloride had a molar concentration of 20 mM, Tween 20 had a volume percentage of 2%, Triton X-100 had a volume percentage of 0.1%, ethyl phenyl polyethylene glycot had a volume percentage of 3%, potassium chloride had a mass concentration of 5 mg/mL, sodium hydroxide had a mass concentration of 50 mg/mL, betaine had a mass concentration of 0.1 mg/mL, bovine sérum albumin had a mass concentration of 100 mg/mL, protéinase K had a mass concentration of 0.02 mg/mL, and lithium dodecyl sulfate had a mass concentration of 30 mg/mL.
Comparative Examples 51-75: The above 25 samples were treated by the magnetic bead method, and real-time fluorescent quantitative PCR amplification was performed.
Ct values in the Examples and Comparative Examples are shown in Table 2. PCR amplification was performed at following conditions:
UDG enzyme reaction at 50°C for 2 min;
thermal dénaturation at 94°C for 3 min;
cydes of amplification at 94°C for 10 sec followed by 60°C for 20 sec; extension and fluorescence collection at 75°C for 20 sec;
melting curve: 62°C to 75°C.
Table 2
| Comparative Examples 51 to 63 | Examples 26 to 38 | Comparative Examples 64 to 75 | Examples 39 to 50 | ||
| Sample 1 | 35.36 | 31.34 | Sample 14 | 23.35 | 24.14 |
| Sample 2 | 29.47 | 24.54 | Sample 15 | 36.03 | 32.69 |
| Sample 3 | 30.18 | 31.1 | Sample 16 | 36.2 | 35.99 |
| Sample 4 | 37.23 | 36.56 | Sample 17 | 36.02 | 35.95 |
| Sample 5 | 33.21 | 28.44 | Sample 18 | 33.44 | 33.2 |
| Sample 6 | 27.94 | 25.69 | Sample 19 | 38.34 | 38.32 |
| Sample 7 | 26.58 | 24.945 | Sample 20 | 30.76 | 29.145 |
| Sample 8 | 31.19 | 30.495 | Sample 21 | 36.49 | 30.85 |
| Sample 9 | 33.64 | 29.675 | Sample 22 | 26.22 | 25.925 |
| Sampîe 10 | 37.95 | 34.455 | Sample 23 | 23.01 | 22.96 |
| Sample 11 | 32.68 | 24.13 | Sample 24 | 31.39 | 27.32 |
| Sample 12 | 27.58 | 23.355 | Sample 25 | 29.26 | 24.015 |
| Sample 13 | 34.14 | 32.605 |
According to the test results, sampies that were positive for respiratory bacteria can be detected in ail comparative examples and examples with a consistent rate of results of io 100% and good accuracy. In addition, a smaller Ct value indicates higher détection sensitivity. Comparing the Ct values, it can be seen that in the examples using the nucleic acid reiease agent and the method for PCR amplification of the nucleic- acid of the présent disclosure, the sensitivity of the multiple amplification and détection of bacteria was équivalent to that using the magnetic bead method. It also shows that the nucleic acid 15 reiease agent and the method for PCR amplification of the nucleic acid of the présent disclosure are not only applicable to amplification and détection of RNA sampies, but also can be applied to amplification and détection of DNA sampies.
3. Détection for HCV viruses
Examples 51 to 70: 20 HCV sérum sampies were prepared. 15 pL of each sample was taken, and 5 pu of a nucleic acid reïease agent was added thereto, followed by standing for 10 min. 30 pL of a PCR reaction solution was added with mixing for performing realtime fluorescence quantitative PCR amplification. The amplification cun/e is shown in FIG. 3. The nucleic acid release agent used comprised Tris-HCI, sodium chloride, potassium chloride, Tween 20,. Triton X-100, ethyl phenyl polyethylene glycol, betaine, bovine sérum albumin, and sodium hydroxide; wherein Tris-HCI had a molar concentration of 200 mM, sodium chloride had a molar concentration of 250 mM, Tween 20 had a volume percentage of 1%, Triton X-100 had a volume percentage of 2%, ethyl phenyl polyethylene glycol had a volume percentage of 2%, potassium chloride had a mass concentration of 7 mg/mL, sodium hydroxide had a mass concentration of 25 mg/mL, betaine had a mass concentration of 10 mg/mL, and bovine sérum albumin had a mass concentration of 60 mg/mL.
Examples 91 to 110: the same HCV sérum sampies as in Examples 51 to 70 were used. 15 pL of each sample was taken, and 5 pL of a nucleic acid release agent was added thereto, followed by standing for 10 min. 30 pL of a PCR reaction solution was added with mixing for performing real-time fluorescence quantitative PCR amplification. The nucleic acid release agent is the same as the nucleic acid release agent used in Examples 1 to 25. Ct values in the examples are shown in Table 3. PCR amplification was performed at following conditions:
pre-denaturation and enzyme activation at 95°C for 1 min;
reverse transcription at 60°C for 30 min;
thermal dénaturation at 95°C for 1 min;
cycles of amplification at 95°C for 15 sec followed by 60°C for 30 sec;
cooling at 25°C for 10 sec.
Table 3
| Examples 91 to 100 | Exampies 51 to 60 | Examples 101 to 110 | Examples 61 to 70 | ||
| Sample 1 | 30.25 | 31.32 | Sample 11 | 24.56 | 25.13 |
| Sample 2 | 27.66 | 29.78 | Sample 12 | 25.60 | 26.03 |
| Sample 3 | 28.31 | 30.28 | Sample 13 | 27.06 | 27.92 |
| Sample 4 | 29.00 | 31.23 | Sample 14 | 27.95 | 28.88 |
| Sample 5 | 28.63 | 31.81 | Sample 15 | 31.05 | 31.64 |
| Sample 6 | 27.25 | 27.94 | Sample 16 | 28.60 | 30.55 |
| Sample 7 | 27.21 | 26.85 | Sample 17 | 29.67 | 30.76 |
| Sampie 8 | 29.29 | 31.27 | Sample 18 | 30.44 | 31.49 |
| Sample 9 | 28.63 | 31.64 | Sample 19 | 19.78 | 20.25 |
| Sample 10 | 30.55 | 31.95 | Sampie 20 | 33.20 | 32.01 |
According to FIG. 3, it can be seen that in sampies that were positive for HCV can be detected in ail exampies using the nucieic acid release agent and the method for PCR amplification of the nucleic acid of the présent disclosure with good accuracy. In addition, it can be seen from Table 3 that the détection sensitivity in Examples 51 to 70 is slightly worse than that in Examples 91 to 100, indicating that the effect of the nucleic acid release agent used in Exampies 51 to 70 is slightly inferior to that of the nucleic acid release agent used in Exampies 91 to 100.
4. Détection for respiratory viruses
Exampies 71 to 90; 20 respiratory virus throat swab sampies (normal saline vehicle) were prepared. 100 pLof each sample was taken and centrifugated at 12000 rpm/min for 10 min, with the supernatant discarded. After adding 50 pL of a nucieic acid release agent and standing for 10 min, 10 pL of the nucieic acid release agent-treated sample was mixed with 40 pL of a PCR réaction solution for performing real-time fluorescence quantitative PCR amplification. The amplification curve is shown in FIG. 1. The nucleic acid release agent used comprised Tris-HCI, sodium chloride, potassium chloride, Tween 20, Triton X-100, ethyi phenyl polyethylene glycol, and sodium hydroxide: wherein Tris-HCI had a molar concentration of 400 mM, sodium chloride had a molar concentration of 150 mM, Tween 20 had a volume percentage of 0.8%, Triton X-100 had a volume percentage of 1.2%, ethyi phenyl polyethylene giycoi had a volume percentage of 1.5%, potassium chloride had a mass concentration of 6 mg/mL, and sodium hydroxide had a mass concentration of 15 rng/mL.
Examples 111 to 130; the same respiratory virus throat swab sampies as in Examples 71 to 90 were used. 100 pL of each sample was taken and centrifugated at 12000 rpm/min for 10min, with the supernatant discarded. After adding 50 pL of a nucleic acid release agent and standing for 10 min, 10 pL of the nucleic acid release agent-treated sample was mixed with 40 pL of a PCR reaction solution for performing real-time fluorescence quantitative PCR amplification. The nucleic acid release agent was the same as the nucleic acid release agent used in Examples 51 to 70.
Ct values in the examples are shown in Table 4. PCR amplification was performed at following conditions:
reverse transcription at 50°C for 30 min;
thermal dénaturation at 95°C for 1 min;
cycles of amplification at 95°C for 15 sec foilowed by 60°C for 30 sec; cooling at 25°C for 10 sec.
Table 4
| Examples 111 to 120 | Examples 71 to 80 | Examples 121 to 130 | Examples 81 to 90 | ||
| Sample 1 | 23.64 | 26.05 | Sample 11 | 27.56 | 28.69 |
| Sample 2 | 25.97 | 28.77 | Sample 12 | 28.67 | 29.61 |
| Sample 3 | 29.55 | 31.28 | Sample 13 | 26.22 | 28.32 |
| Sample 4 | 30.52 | 32.65 | Sample 14 | 27.88 | 29.78 |
| Sample 5 | 28.10 | 28.55 | Sample 15 | 31.36 | 31.54 |
| Sample 6 | 31.69 | 34.12 | Sample 16 | 30.69 | 33.69 |
| Sample 7 | 25.19 | 26.12 | Sample 17 | 29.89 | 29.47 |
| Sample 8 | 30.26 | 32.36 | Sample 18 | 30.79 | 32.34 |
| Sample 9 | 28.96 | 31.59 | Sample 19 | 29.20 | 32.69 |
| Sample 10 | 31.60 | 31.35 | Sample 20 | 33.59 | 33.06 |
According to FIG. 4, it can be seen that samples that were positive for respiratory virus can be detected in ail examples using the nucleic acid release agent and the method for PCR amplification of the nucleic acid of the présent disclosure with good accuracy. In addition, it can be seen from Table 4 that the détection sensitivity in Examples 71 to 90 was slightly worse than that in Examples 111 to 130, indicating that the effectof the nucleic acid release agent used in Examples 71 to 90 was slightly inferior to that of the nucleic acid release agent used in Examples 111 to 130.
The technical features of the above-described embodiments may be combined arbitrarily. To simplify the description, not ail of the possible combinations of the technical features in the above embodiments are described. However, ail of the combinations of these technical features should be considered as within the scope of the présent disclosure, as long as such combinations do not contradict with each other.
The above-described embodiments merely represent several embodiments of the présent disclosure, and the description thereof is more spécifie and detailed, but it should not be construed as limiting the scope of the présent disclosure. It should be noted that, for those skilled in the art, several variations and improvements may be made without departing from the concept of the présent disclosure, and these are ail within the protection scope of the présent disclosure. Therefore, the scope of the présent disclosure shall be defined by the appended claims.
Claims (10)
1. A nucleic acid release agent, comprising Tris-HCI, sodium chloride, potassium chloride, Tween 20, Triton X-100, ethyl phenyl polyethylene glycol and a strong base; wherein Tris-HCI has a molar concentration ranging from 0.5 mM to 500 mM, sodium chloride has a molar concentration from 20 mM to 500 mM, potassium chloride has a mass concentration ranging from 5mg/mL to 8mg/mL, Tween 20 has a volume percentage ranging from 0.1% to 2%, Triton X-100 has a volume percentage ranging from 0.1% to 3%, ethyl phenyl polyethylene giycol has a volume percentage ranging from 0.1% to 3%, and the strong base has a mass concentration ranging from 2 mg/mL to 50 mg/mL.
2. The nucleic acid release agent according to claim 1, further comprising betaine and bovine sérum albumin, wherein betaine has a mass concentration ranging from 0.1 mg/mL to 20 mg/mL, and bovine sérum albumin has a mass concentration ranging from 5 mg/mL to 100 mg/mL.
3. The nucleic acid release agent according to claim 1, further comprising protéinase K and lithium dodecyl sulfate, wherein protéinase K has a mass concentration ranging from 0.02 mg/mL to 1.5 mg/mL, and lithium dodecyl sulfate has a mass concentration ranging from 0.4 mg/mL to 30 mg/mL.
4. A method for PCR amplification of a nucleic acid, comprising steps of: mixing the nucleic acid release agent according to any one of claims 1 to 3 with a sample, placing the mixture at 25°C to 60°C for 2 min to 10 min, and adding a PCR reaction solution for PCR amplification.
5. The method according to claim 4, further comprising a sample pretreatment step of mixing the sample with polyethylene glycol foilowed by centrifugation to collect a precipitate before mixing the nucleic acid release agent with the sample.
6. The method according to claim 4, wherein the PCR amplification of intestinal viruses in the sample is performed at following conditions:
reverse transcription at 48°C to 52°C for 28 min to 32 min;
thermal dénaturation at 93°C to 97°C for 0.9 min to 1.1 min;
several cycles of amplification at 93°C to 97°C for 13 sec to 17 sec followed by 53°C to 57°C for 28 sec to 32 sec;
cooling at 23°C to 27°C for 8 sec to 12 sec.
7. The method according to claim 4, wherein the PCR amplification of hepatitis C virus in the sample is performed at following conditions:
pre-denaturation and enzyme activation at 93°C to 97°C for 0.9 min to 1.1 min;
reverse transcription at 58°C to 62°C for 28 min to 32 min;
thermal dénaturation at 93°C to 97°C for 0.9 min to 1.1 min;
several cycles of amplification at 93°C to 97°C for 13 sec to 17 sec followed by
58°C to 62 °C for 28 sec to 32 sec;
cooling at 23°C to 27°C for 8 sec to 12 sec.
8. The method according to claim 4, wherein the PCR amplification of respiratory viruses in the sample is performed at following conditions: reverse transcription at 48°C to 52°C for 28 min to 32 min;
thermal dénaturation at 93°C to 97°C for 0.9 min to 1.1 min;
several cycles of amplification at 93°C to 97°C for 13 sec to 17 sec followed by 58°C to 62°C for 28 sec to 32 sec;
cooling at 23°C to 27°C for 8 sec to 12 sec.
9. The method according to claim 4, wherein the PCR amplification of respiratory bacteria in the sample is performed at following conditions: UDG enzyme reaction at 48°C to 52°C for 1.9 min to 2.1 min;
thermal dénaturation at 92°C to 96°C for 2.9 min to 3.1 min;
several cycles of amplification at 92°C to 96°C for 8 sec to 12 sec followed by 58°C to 62°C for 18 sec to 22 sec;
extension and fluorescence collection at 73°C to 77°C for 18 secto 22 sec;
melting curve: 62QC to 75°C.
10. A PCR amplification kit, comprising the nucleic acid reiease agent according to any one of claims 1 to 3 and a PCR reaction solution.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910014324.3 | 2019-01-08 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| OA20437A true OA20437A (en) | 2022-08-08 |
Family
ID=
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP3910067B1 (en) | Nucleic acid release agent, nucleic acid pcr amplification method and pcr amplification kit | |
| JP6750178B2 (en) | Stabilization of biological samples | |
| Aebischer et al. | Rapid genome detection of Schmallenberg virus and bovine viral diarrhea virus by use of isothermal amplification methods and high-speed real-time reverse transcriptase PCR | |
| CN107475252B (en) | Nucleic acid releasing agent, method for rapidly releasing nucleic acid and application thereof | |
| CN112921075B (en) | Hand-taking-free reagent for blood sample fluorescence PCR and application thereof | |
| EP0953635B1 (en) | Improved methods for extracting nucleic acids from tissue samples and paraffin-embedded tissues | |
| Batista et al. | Whole genome sequencing of hepatitis A virus using a PCR-free single-molecule nanopore sequencing approach | |
| CN114410836B (en) | Kit and method for detecting human parvovirus B19 by integrating sample treatment, nucleic acid extraction and multiplex isothermal amplification | |
| JP2001352982A (en) | Method for synthesizing nucleic acid | |
| OA20437A (en) | Nucleic acid release agent, nucleic acid PCR amplification method and PCR amplification kit | |
| JP6301260B2 (en) | RNA preparation method | |
| CN113564157B (en) | PCR reaction system, kit and PCR method | |
| US20210207125A1 (en) | Method of isolating nucleic acids for long sequencing reads | |
| CN113621607A (en) | Lysis solution and application thereof | |
| JP2007518425A (en) | Primer and probe design for efficient amplification and detection of HCV 3 'untranslated region | |
| US11021763B2 (en) | Assay for detecting hepatitis C virus (HCV) | |
| CN112063615A (en) | Genome DNA extracting solution, genome DNA extracting method and application thereof | |
| JPWO2008146649A1 (en) | Enzyme-containing gel and nucleic acid amplification kit | |
| WO2023235875A1 (en) | Methods for sample preparation | |
| CN114480573A (en) | DNA direct amplification reagent and application thereof | |
| JP2006187221A (en) | Pretreatment method of whole blood sample and method for amplifying nucleic acid | |
| CN116286714A (en) | Fusion Taq DNA polymerase and preparation method and application thereof | |
| CN116590471A (en) | Detection device and method for detecting early-stage hepatitis B-to-liver cancer based on specific PCR | |
| Liu et al. | Expression of NASG gene and its role in human nasopharyngeal homogenous tissue cells | |
| Horlitz et al. | Yields of viral and circulating cell-free nucleic acids using the QIAamp® circulating nucleic acid kit |