AU2021103963A4 - Paper-based chromatographic viral nucleic acid extraction kit - Google Patents
Paper-based chromatographic viral nucleic acid extraction kit Download PDFInfo
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- AU2021103963A4 AU2021103963A4 AU2021103963A AU2021103963A AU2021103963A4 AU 2021103963 A4 AU2021103963 A4 AU 2021103963A4 AU 2021103963 A AU2021103963 A AU 2021103963A AU 2021103963 A AU2021103963 A AU 2021103963A AU 2021103963 A4 AU2021103963 A4 AU 2021103963A4
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- 238000000605 extraction Methods 0.000 title claims abstract description 71
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 60
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 60
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 60
- 230000003612 virological effect Effects 0.000 title claims abstract description 40
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 24
- 238000004140 cleaning Methods 0.000 claims abstract description 22
- 239000003480 eluent Substances 0.000 claims abstract description 13
- 239000012139 lysis buffer Substances 0.000 claims abstract description 13
- 229920000742 Cotton Polymers 0.000 claims abstract description 10
- 239000000203 mixture Substances 0.000 claims abstract description 8
- 210000002966 serum Anatomy 0.000 claims description 15
- 239000000243 solution Substances 0.000 claims description 14
- 239000007983 Tris buffer Substances 0.000 claims description 9
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 9
- 230000002934 lysing effect Effects 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 6
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 6
- 241000700605 Viruses Species 0.000 claims description 5
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 3
- 108010067770 Endopeptidase K Proteins 0.000 claims description 3
- 238000010521 absorption reaction Methods 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 229960000789 guanidine hydrochloride Drugs 0.000 claims description 3
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 238000000034 method Methods 0.000 abstract description 22
- 238000001514 detection method Methods 0.000 abstract description 7
- 238000012360 testing method Methods 0.000 abstract description 5
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 4
- 238000012123 point-of-care testing Methods 0.000 abstract description 4
- 238000000746 purification Methods 0.000 abstract description 3
- 238000009472 formulation Methods 0.000 abstract description 2
- 238000003757 reverse transcription PCR Methods 0.000 abstract description 2
- 239000000523 sample Substances 0.000 description 16
- 238000011529 RT qPCR Methods 0.000 description 5
- 238000003762 quantitative reverse transcription PCR Methods 0.000 description 5
- 238000009835 boiling Methods 0.000 description 4
- 230000009089 cytolysis Effects 0.000 description 4
- 230000005298 paramagnetic effect Effects 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 238000007796 conventional method Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 239000002253 acid Substances 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 230000005291 magnetic effect Effects 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 102000011931 Nucleoproteins Human genes 0.000 description 1
- 108010061100 Nucleoproteins Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 241001112090 Pseudovirus Species 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012501 chromatography medium Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M1/00—Apparatus for enzymology or microbiology
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/101—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/34—Purifying; Cleaning
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
- G01N2030/8827—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving nucleic acids
Abstract
The present disclosure discloses a paper-based chromatographic viral nucleic
acid extraction kit, and relates to the technical field of nucleic acid extraction. The
paper-based chromatographic viral nucleic acid extraction kit includes the following
components: a lysis buffer, a cleaning solution, an eluent, and a chromatographic
extraction tube, wherein the chromatographic extraction tube is a 1.5 to 2.0 mL EP
tube additionally provided with a chromatography paper sheet and a chromatography
cotton. According to the paper-based chromatographic viral nucleic acid extraction kit,
formulations of the reagents are optimized based on a sample paper-based
chromatographic theory, trace amount of viral nucleic acid can be quickly captured
from 200 pL of sample, the operating process is very simple, the extraction and
purification of viral nucleic acid can be finished within 10 min, which saves a lot of
time and reagents, and the extraction efficiency is significantly improved. Moreover,
no expensive instrument is required, compared with that of the existing methods, the
test cost is significantly reduced, and thus it is suitable for Point of Care Testing
(POCT). The extraction method has the advantages of rapidness, high efficiency,
simple operation, etc., and fully meets requirements of subsequent molecular
detection such as PCR or RT-PCR, which saves a lot of time for testing, diagnosing
and research staffs.
- 10-
mpI.ncauon P0t
525000
$00.000
475,000
450.000
425-000
400.000
375.000
350.000
325 000
300,000
275000
250000
225000, The present
200 000 extraction method Control extraction
175000 method
150000
125000
100000
75000
50-000
25000
0 .99=
-25.000,
Cycle
Fig. 1
- 11-
Description
mpI.ncauon P0t 525000 $00.000 475,000 450.000
425-000
400.000 375.000
350.000 325 000
300,000
275000
250000
225000, The present 200 000 extraction method Control extraction 175000 method 150000 125000 100000 75000 50-000 25000 0 .99=
-25.000,
Cycle
Fig. 1
The present disclosure relates to the technical field of nucleic acid extraction,
and particularly relates to a paper-based chromatographic viral nucleic acid extraction
kit.
Nucleic acid is a biomacromolecular compound which is formed by
polymerization of a plurality of nucleotides, and is one of the most basic substances of
life. Nucleic acids are widely found in all animal and plant cells, and microorganisms.
Nucleic acids in organisms often combine with proteins to form nucleoproteins.
Different nucleic acids differ in their chemical composition, nucleotide sequences, etc.
Efficient and simple extraction of target nucleic acids from complex biological
materials is the key to molecular detection and diagnostic technology. Currently, the
purification technologies of viral nucleic acid are mainly divided into three types,
which are a conventional method, a column membrane method and a paramagnetic
particle method, respectively.
Among the existing nucleic acid extraction methods, the conventional method
includes a commonly-used boiling method and a TRIZOL lysis method. Nucleic acid
extracted by the boiling method has relatively low purity, the TRIZOL lysis method is
complicated and time-consuming, the column membrane method is not conductive to
automatic operation and is also time-consuming, the paramagnetic particle method
uses magnetic beads to absorb nucleic acid, but impurities such as impure proteins are
easily absorbed at the same time, so it is difficult to obtain high-purity nucleic acid,
and the cost is high. Therefore, there is room for improvement in the nucleic acid
extraction methods. For this purpose, a paper-based chromatographic viral nucleic
acid extraction kit is proposed.
In view of the shortcomings of the prior art, the present disclosure provides a
paper-based chromatographic viral nucleic acid extraction kit, which solves the
problems mentioned in the above background that among the existing nucleic acid
extraction methods, a conventional method includes a commonly-used boiling method
and a TRIZOL lysis method, nucleic acid extracted by the boiling method has
relatively low purity, the TRIZOL lysis method is complicated and time-consuming,
the column membrane method is not conductive to automatic operation and is also
time-consuming, the paramagnetic particle method uses magnetic beads to absorb
nucleic acid, but impurities such as impure proteins are easily absorbed at the same
time, so it is difficult to obtain high-purity nucleic acid, and the cost is high, and
therefore, there is room for improvement in the nucleic acid extraction methods.
To achieve the above objective, the present disclosure provides the following
technical solution: a paper-based chromatographic viral nucleic acid extraction kit
including the following components: a lysis buffer, a cleaning solution, an eluent, and
a chromatographic extraction tube, wherein the chromatographic extraction tube is a
1.5 to 2.0 mL EP tube additionally provided with a chromatography paper sheet and a
chromatography cotton.
Optionally, a nucleic acid extraction method of the paper-based chromatographic
viral nucleic acid extraction kit includes the following specific steps:
(1) lysing
lysing a virus in a serum: first adding 200 pL of serum sample into a 1.5 to 2 mL
centrifuge tube, then adding 500 pL of lysis buffer into the tube, and uniformly
mixing the mixture by reversing the tube at room temperature for 1 to 3 min;
(2) absorbing
dropwise adding 700 pL of lysed sample obtained at the previous step to a center
of the paper-based chromatography paper sheet in the chromatographic extraction
tube to allow the lysed sample to be absorbed completely;
(3) cleaning dropwise adding 200 pL of cleaning solution into the chromatographic extraction tube obtained at the previous step for cleaning;
(4) eluting
transferring the chromatography paper sheet obtained at the previous step into a
new 1.5 to 2.0 mL EP tube, and then adding 50 pL of eluent or DEPC water into the
tube to obtain viral nucleic acid.
Optionally, the lysis buffer contains the following components at a concentration:
I to 1.5 M guanidine hydrochloride, 30 to 50 mM Tris (pH 8.0), 50 to 100 mM NaCl,
to 10 mM EDTA, 1% Tween-20 (v/v), and 40 g/ml proteinase K.
Optionally, the cleaning solution contains 10 to 15 mM Tris (pH 8.0) and 0.1%
Tween-20 (v/v).
Optionally, the eluent is 10 to 15 mM/L Tris (pH 8.0) DEPC aqueous solution.
Optionally, the chromatography paper sheet is a circular paper sheet having a
diameter of 6 mm and a thickness of 3 mm.
Optionally, the chromatography cotton is a cylindrical cotton having a diameter
of 9 mm, a length of 25 mm, and a water absorption of 100 to 150%.
The present disclosure provides a paper-based chromatographic viral nucleic
acid extraction kit which has the following beneficial effects: according to the
paper-based chromatographic viral nucleic acid extraction kit, formulations of the
reagents are optimized based on a sample paper-based chromatographic theory, trace
amount of viral nucleic acid can be quickly captured from 200 pL of sample, the
operating process is very simple, the extraction and purification of viral nucleic acid
can be finished within 10 min, which saves a lot of time and reagents, and the
extraction efficiency is significantly improved, and moreover, no expensive
instrument is required, and compared with that of the existing methods, the test cost is
significantly reduced, and thus it is suitable for Point of Care Testing (POCT).
The extraction method of the paper-based chromatographic viral nucleic acid
extraction kit has the advantages of rapidness, high efficiency, simple operation, etc.,
and fully meets requirements of subsequent molecular detection such as PCR or
RT-PCR, which saves a lot of time for testing, diagnosing and research staffs.
Moreover, the viral extraction efficiency can be improved, and at the same time, the threshold of hardware conditions for operating this technology is lowered. The present disclosure adopts a unique and simple paper-based chromatography medium, and an alcohol-free lysis buffer and eluent, obtains high-quality viral nucleic acid molecules (DNA or RNA) by a three-step method, can be applied to an automatic platform, and has a wider application prospect.
BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a diagram of amplification curves of HEV pseudovirus nucleic acid samples extracted by the present extraction method and a control extraction method; Fig. 2 is a table of sequences of primers and probes of RT-qPCR according to the present disclosure; Fig. 3 is a table of reaction system and routine of RT-qPCR according to the present disclosure; and Fig. 4 is a table of average Ct values of RT-qPCR detection according to the present disclosure.
DETAILED DESCRIPTION The technical solutions in embodiments of the present disclosure will be clearly and completely described below in conjunction with specific embodiments of the present disclosure. Obviously, the described embodiments are not all embodiments but part of embodiments of the present disclosure. A paper-based chromatographic viral nucleic acid extraction kit includes the following components: a lysis buffer, a cleaning solution, an eluent, and a chromatographic extraction tube, wherein the chromatographic extraction tube is a 1.5 to 2.0 mL EP tube additionally provided with a chromatography paper sheet and a chromatography cotton. The lysis buffer contains the following components at a concentration: 1 to 1.5 M guanidine hydrochloride, 30 to 50 mM Tris (pH 8.0), 50 to 100 mM NaCl, 5 to 10 mM EDTA, 1% Tween-20 (v/v), and 40 g/ml proteinase K; the cleaning solution contains 10 to 15 mM Tris (pH 8.0) and 0.1% Tween-20 (v/v); the eluent is 10 to 15 mM/L Tris (pH 8.0) DEPC aqueous solution; the chromatography paper sheet is a circular paper sheet having a diameter of 6 mm and a thickness of 3 mm; and the chromatography cotton is a cylindrical cotton having a diameter of 9 mm, a length of mm, and a water absorption of 100 to 150%.
A nucleic acid extraction method of the paper-based chromatographic viral
nucleic acid extraction kit includes the following specific steps:
(1) lysing
a virus in a serum is lysed: first 200 pL of serum sample is added into a 1.5 to 2
mL centrifuge tube, then 500 pL of lysis buffer is added into the tube, and the mixture
is uniformly mixed by reversing the tube at room temperature for I to 3 min;
(2) absorbing
700 pL of lysed sample obtained at the previous step is dropwise added to a
center of the paper-based chromatography paper sheet in the chromatographic
extraction tube to allow the lysed sample to be absorbed completely;
(3) cleaning
200 pL of cleaning solution is dropwise added into the chromatographic
extraction tube obtained at the previous step for cleaning;
(4) eluting
the chromatography paper sheet obtained at the previous step is transferred into a
new 1.5 to 2.0 mL EP tube, and then 50 pL of eluent or DEPC water is added into the
tube to obtain viral nucleic acid.
In conclusion, according to the paper-based chromatographic viral nucleic acid
extraction kit, when used, the paper-based chromatographic viral nucleic acid
extraction kit includes the following specific steps:
(1) lysing
a virus in a serum is lysed: first 200 pL of serum sample is added into a 1.5 to 2
mL centrifuge tube, then 500 pL of lysis buffer is added into the tube, and the mixture
is uniformly mixed by reversing the tube at room temperature for I to 3 min;
(2) absorbing
700 pL of lysed sample obtained at the previous step is dropwise added to a center of the paper-based chromatography paper sheet in the chromatographic extraction tube to allow the lysed sample to be absorbed completely; (3) cleaning 200 pL of cleaning solution is dropwise added into the chromatographic extraction tube obtained at the previous step for cleaning; (4) eluting the chromatography paper sheet obtained at the previous step is transferred into a new 1.5 to 2.0 mL EP tube, and then 50 pL of eluent or DEPC water is added into the tube to obtain the viral nucleic acid. Embodiment 1 The present disclosure was compared with a paramagnetic particle method kit manufactured by a domestic manufacturer in terms of detection of HEV in a serum. First, imitated samples were prepared: three positive serums and three negative serums, each of 200 pL, were added into 1.5 ml centrifuge tubes, respectively, wherein the positive serums were respectively added with a WHO HEV standard substance at a final concentration of 10 IU, and the negative serums were not added with the WHO HEV standard substance. (1) lysing a virus in the serum was lysed: 500 pL of lysis buffer was added into each tube containing the imitated sample, and the mixture was uniformly mixed by reversing the tube at room temperature for 1 to 3 min; (2) absorbing the lysed sample in each tube obtained at the previous step was dropwise added to a center of the paper-based chromatography paper sheet in the chromatographic extraction tube to allow the lysed sample to be absorbed completely; (3) cleaning 200 pL of cleaning solution was dropwise added into each chromatographic extraction tube obtained at the previous step for cleaning; (4) eluting each chromatography paper sheet obtained at the previous step was transferred into a new 1.5 mL EP tube, and then 50 pL of eluent was added into each tube to obtain viral nucleic acid.
Then, PCR amplification was performed on the viral nucleic acid by using
primers and probes shown in Fig. 2, and RT-qPCR reaction systems and routines
shown in Fig. 3, and extraction effects of the kits were evaluated by using Ct values of
detection. Average Ct values of RT-qPCR detection obtained in tests are shown in Fig.
4. It can be seen from Fig. 1 to Fig. 4 that the concentration of the HEV nucleic acid
extracted by the present extraction method (the kit of the present disclosure) is higher
than that of the HEV nucleic acid extracted by the kit of the control extraction method
(because the CT value is smaller), and the repeatability is approximate (all CV values
<5%). Therefore, compared with the control kit, the paper-based chromatographic
viral nucleic acid extraction kit of the present disclosure has higher efficiency on
extraction of viral nucleic acid (HEV) in a serum.
The above is only the preferable specific embodiments of the present disclosure
and not intended to limit the scope of protection of the present disclosure. Any
equivalent replacement or variation apparent to those of ordinary skill in the art within
the technical scope disclosed by the present disclosure based on the technical solution
and inventive concept of the present disclosure shall fall within the scope of
protection of the present disclosure.
Claims (7)
1. A paper-based chromatographic viral nucleic acid extraction kit comprising
the following components: a lysis buffer, a cleaning solution, an eluent, and a
chromatographic extraction tube, wherein the chromatographic extraction tube is a 1.5
to 2.0 mL EP tube additionally provided with a chromatography paper sheet and a
chromatography cotton.
2. The paper-based chromatographic viral nucleic acid extraction kit of claim
1, wherein a nucleic acid extraction method of the paper-based chromatographic viral
nucleic acid extraction kit comprises the following specific steps:
(1) lysing
lysing a virus in a serum: first adding 200 pL of serum sample into a 1.5 to 2
mL centrifuge tube, then adding 500 pL of lysis buffer into the tube, and uniformly
mixing the mixture by reversing the tube at room temperature for 1 to 3 min;
(2) absorbing
dropwise adding 700 pL of lysed sample obtained at the previous step to a
center of the paper-based chromatography paper sheet in the chromatographic
extraction tube to allow the lysed sample to be absorbed completely;
(3) cleaning
dropwise adding 200 pL of cleaning solution into the chromatographic
extraction tube obtained at the previous step for cleaning;
(4) eluting
transferring the chromatography paper sheet obtained at the previous step into
a new 1.5 to 2.0 mL EP tube, and then adding 50 pL of eluent or DEPC water into the
tube to obtain viral nucleic acid.
3. The paper-based chromatographic viral nucleic acid extraction kit of claim
1, wherein the lysis buffer comprises the following components at a concentration: 1 to 1.5 M guanidine hydrochloride, 30 to 50 mM Tris (pH 8.0), 50 to 100 mM NaCl, 5 to 10 mM EDTA, 1% Tween-20 (v/v), and 40 g/ml proteinase K.
4. The paper-based chromatographic viral nucleic acid extraction kit of claim
1, wherein the cleaning solution comprises 10 to 15 mM Tris (pH 8.0) and 0.1%
Tween-20 (v/v).
5. The paper-based chromatographic viral nucleic acid extraction kit of claim
1, wherein the eluent is 10 to 15 mM/L Tris (pH 8.0) DEPC aqueous solution.
6. The paper-based chromatographic viral nucleic acid extraction kit of claim
1, wherein the chromatography paper sheet is a circular paper sheet having a diameter
of 6 mm and a thickness of 3 mm.
7. The paper-based chromatographic viral nucleic acid extraction kit of claim
1, wherein the chromatography cotton is a cylindrical cotton having a diameter of 9
mm, a length of 25 mm, and a water absorption of 100 to 150%.
Fig. 1
Fig. 2
Fig. 3
Fig. 4
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2021103963A AU2021103963A4 (en) | 2021-07-08 | 2021-07-08 | Paper-based chromatographic viral nucleic acid extraction kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2021103963A AU2021103963A4 (en) | 2021-07-08 | 2021-07-08 | Paper-based chromatographic viral nucleic acid extraction kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| AU2021103963A4 true AU2021103963A4 (en) | 2021-09-09 |
Family
ID=77563712
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU2021103963A Ceased AU2021103963A4 (en) | 2021-07-08 | 2021-07-08 | Paper-based chromatographic viral nucleic acid extraction kit |
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| Country | Link |
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2021
- 2021-07-08 AU AU2021103963A patent/AU2021103963A4/en not_active Ceased
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