CN118028436B - A lysate and a kit and method for detecting nucleic acid in a biological sample - Google Patents
A lysate and a kit and method for detecting nucleic acid in a biological sample Download PDFInfo
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Abstract
The invention provides a lysate and a kit and a method for detecting nucleic acid in a biological sample, which improve the detection sensitivity and detection rate of nucleic acid in a nucleic acid sample containing polysaccharide components and a viscous biological sample in LAMP amplification by adding a proper amount of Sr 2+ into the lysate, thereby being beneficial to improving the detection efficiency of nucleic acid in the nucleic acid sample containing polysaccharide components or the viscous biological sample.
Description
Technical Field
The disclosure relates to the technical field of nucleic acid detection, in particular to a lysate, a kit and a method for detecting nucleic acid in a biological sample.
Background
Loop-mediated isothermal amplification (LAMP) technology, based on strand-displacement DNA polymerase and special primer design, allows multiple rounds of amplification at constant temperature (typically 60-65 ℃) to produce large amounts of target DNA in a short period of time. Compared with the conventional PCR, the LAMP has higher sensitivity and amplification efficiency, higher detection accuracy for low-concentration samples, higher specificity because four primers in the LAMP system specifically target six sections of regions in a pre-amplified DNA sequence, no influence of non-target DNA in the amplification process, no need of processes of thermal denaturation, temperature circulation, electrophoresis and the like of a template, no dependence on a precise thermal cycler and other complex instrument equipment, great cost and time saving, easy popularization and application in a base layer with deficient resources, and suitability for on-site rapid detection.
At present, the LAMP technology is widely used in the fields of biological medicine, environment, water quality monitoring, food safety and the like as a rapid, sensitive and high-specificity nucleic acid amplification method. In LAMP detection, DNA is not required to be extracted specially, and cell lysate is directly utilized for amplification and detection, however, in the detection process of viscous biological samples (such as oral swabs), the presence of polysaccharide components can inhibit the LAMP amplification process, so that the sensitivity and detection rate of LAMP detection are reduced. Therefore, there is a need to develop a method capable of improving the detection efficiency of nucleic acids in a viscous biological sample.
Disclosure of Invention
In order to achieve the above object, the present disclosure provides the following technical solutions:
In one aspect, the disclosure provides a method for detecting nucleic acid in a biological sample, comprising (1) releasing nucleic acid in a viscous biological sample or a polysaccharide sample by using a lysate to obtain a sample lysate, and (2) sampling the lysate to perform LAMP amplification reaction, wherein the lysate contains Sr 2+. In some embodiments, the biological sample is a biological sample comprising a polysaccharide. In some embodiments, further, the sample is a viscous biological sample. In some embodiments, the sample is an oral swab biological sample. In some embodiments, the polysaccharide comprises dextran sulfate. In some embodiments, the concentration of target nucleic acid in the sample lysate is selected from 0.02-2 pg/μl, preferably from 0.05 pg/μL、0.1 pg/μL、0.2 pg/μL、0.3 pg/μL、0.4 pg/μL、0.5 pg/μL、0.6 pg/μL、0.7 pg/μL、0.8 pg/μL、0.9 pg/μL、1.0 pg/μL、1.1 pg/μL、1.2 pg/μL、1.3 pg/μL、1.4 pg/μL、1.5 pg/μL、1.6 pg/μL、1.7 pg/μL、1.8 pg/μL、1.9 pg/μL.
In some embodiments, the concentration of Sr 2+ in the lysate may be selected from 0.5-100 mM, preferably 0.5-50 mM, more preferably 0.5-25 mM. In some embodiments, the concentration of Sr 2+ may be selected from 0.5 mM, 1mM, 5 mM, 10 mM, 15 mM, 20 mM, or 25 mM. In some embodiments, sr 2+ in the lysate is produced after addition of SrCl 2.
In some embodiments, the lysate further comprises a buffer component, a surfactant, a stabilizer, a chelating agent, and the like.
In some embodiments, the buffer component is Tris, the concentration of Tris being 5-50 mM, preferably 10-20mM, more preferably 10 mM.
In some embodiments, the surfactant includes, but is not limited to, at least one of tween 20, triton x-100, sodium Dodecyl Sulfate (SDS), sodium dodecyl sulfate, sodium dodecyl sarcosinate, lithium dodecyl sulfate. Preferably SDS, the mass fraction of SDS is 0.5-10%, preferably 1-5%, more preferably 1%.
In some embodiments, the stabilizing agent comprises a nonionic surfactant and/or an amino acid, wherein the nonionic surfactant may be selected from PEG200, PEG2000, PEG400, PEG4000, PEG800, PEG8000, etc., and the amino acid may be selected from glycine, lysine, etc. In some embodiments, the chelating agent may be selected from at least one of EGTA, EDTA-2 Na, EDTA-4 Na, EDTA, sodium citrate, EGTA, HEDTA. Preferably the nonionic surfactant PEG200, the mass fraction of said PEG200 is 1-10%, preferably 1-5%, more preferably 2%.
In some embodiments, the chelating agent is preferably EDTA, the concentration of EDTA being 0.5-10 mM EDTA, preferably 1-5 mM, more preferably 1 mM.
In some embodiments, the lysate is at a pH of 7.0-9.0, with a preferred lysate pH of 7.0.
In some embodiments, the lysate has a pH of 7.0-9.0 and comprises 5-50 mM Tris, 0.5-10 mM EDTA, 0.5-10 w/v% SDS, 1-10 w/v% PEG200, and 0.5-100 mM Sr 2+. In some embodiments, the lysate is at pH 7.0, comprising 10 mM Tris, 1mM EDTA, 1 w/v% SDS, 2 w/v% PEG200, and 0.5-100 mM Sr 2+. In some embodiments, the concentration of Sr 2+ in the lysate is preferably in the range of 0.5-50 mM, more preferably in the range of 0.5-25 mM. In some embodiments, the concentration of Sr 2+ may be selected from 0.5 mM, 1mM, 5mM, 10 mM, 15 mM, 20 mM, or 25 mM.
In some embodiments, the amplification system of the LAMP amplification reaction comprises a buffer, mgSO 4, dntps, bst DNA polymerase, primer set, and fluorescent indicator. In some embodiments, the final concentration of MgSO 4 is 2-10 mM, the final concentration of dNTPs is 1-2 mM, and the final concentration of Bst DNA polymerase is 0.16-0.48U/. Mu.L in the amplification system. In some embodiments, the amplification system further comprises sterilized enzyme-free water. In some embodiments, the buffer comprises Tris-HCl, KCl, (NH 4)2SO4、MgSO4, and Triton X-100.
In some embodiments, the fluorescent indicator may be selected from hydroxynaphthol blue (HNB), calcein, SYBR Green, evaGreen, syto, and the like. In some embodiments, the fluorescent indicator is preferably Syto.
In some embodiments, the primer set is a specific LAMP primer set designed for a target nucleic acid sequence, comprising a pair of outer primers F3/B3, a pair of inner primers FIP/BIP, and optionally further comprising loop primers LoopF and/or LoopB. In some embodiments, the concentration of the F3/B3 primer pair in the amplification system is 0.1-0.4. Mu.M, and the concentration of the FIP/BIP primer pair is 0.8-3.2. Mu.M. In some embodiments, the concentration of the LoopF and/or LoopB primer pair is 0.4-1.6 μm. Where "concentration of primer pair" refers to the concentration of each primer in the primer pair.
In some embodiments, the amplification system of the LAMP reaction comprises 1 Xbuffer, 8mM MgSO 4, 1.6. Mu.M FIP/BIP primer pair, 0.2. Mu. M F3/B3 primer pair, 0.8. Mu.M LoopB primer, 1.4 mM dNTP, syto dye, and 0.32U/. Mu.L Bst DNA polymerase. In some embodiments, the pH of the 1 Xbuffer is 7.0-9.0, including 10-30 mM Tris-HCl, 50-150 mM KCl, 5-20 mM (NH 4)2SO4、1-5 mM MgSO4 and 1-3 w/v% Triton X-100. In some embodiments, the pH of the 1 Xbuffer is 8.8, including 20 mM Tris-HCl, 100 mM KCl, 10mM (NH 4)2SO4、2 mM MgSO4 and 1 w/v% Triton X-100).
The viscous biological sample described in the present disclosure refers to a sample derived from an organism, which contains a large amount of polysaccharide components. In some embodiments, the viscous biological sample is a sputum, cervical mucus, nasopharyngeal swab, buccal swab, genital swab, lavage, or the like, preferably an buccal swab biological sample.
In some embodiments, the methods described in the present disclosure may be used for detection of a trace nucleic acid component sample. In some embodiments, the concentration of target nucleic acid added to the amplification system is 0.004-0.4 pg/. Mu.L. In some embodiments, the concentration of target nucleic acid added to the amplification system is 0.004-0.4 pg/. Mu.L.
In another aspect, the present disclosure also provides a lysate comprising Sr 2+. In some embodiments, the concentration of Sr 2+ may be selected from 0.5-100 mM, preferably 0.5-50 mM, more preferably 0.5-25 mM. In some embodiments, the concentration of Sr 2+ may be selected from 0.5 mM, 1mM, 5mM, 10 mM, 15 mM, 20mM, or 25 mM. In some embodiments, sr 2+ in the lysate is produced after addition of SrCl 2.
In some embodiments, the lysate is suitable for biological samples containing polysaccharide components. In some embodiments, the polysaccharide comprises dextran sulfate. In some embodiments, the lysate is suitable for use with viscous biological samples, preferably sputum, cervical mucus, nasopharyngeal swab, oral swab, genital swab, lavage, and more preferably oral swab biological samples.
In some embodiments, the lysate further comprises a buffer component, a surfactant, a stabilizer, a chelating agent, and the like.
In some embodiments, the lysate has a pH of 7.0-9.0 and comprises 0.5-50 mM Tris, 0.5-10 mM EDTA, 0.5-10 w/v% SDS, 1-10 w/v% PEG200, and 0.5-100 mM Sr 2+. In some embodiments, the lysate is at pH 7.0, comprising 10mM Tris, 1mM EDTA, 1 w/v% SDS, 2 w/v% PEG200, and 0.5-100 mM Sr 2+. In some embodiments, the concentration of Sr 2+ is preferably 0.5-50 mM, more preferably 0.5-25 mM. In some embodiments, the concentration of Sr 2+ may be selected from 0.5 mM, 1mM, 5mM, 10mM, 15 mM, 20 mM, or 25 mM. In some embodiments, the Sr 2+ is produced after addition of SrCl 2.
In another aspect, the present disclosure also provides a kit for detecting nucleic acid in a biological sample, comprising 1) a sample lysate as described in the present disclosure, and 2) a LAMP amplification system. In some embodiments, the LAMP amplification system comprises one or more buffer schemes, comprising 1 Xbuffer (pH 7.0-9.0, comprising 10-30 mM Tris-HCl, 50-150 mM KCl, 5-20 mM (NH 4)2SO4、1-5 mM MgSO4 and 1-3 w/v% Triton X-100), 8 mM MgSO 4, 1.6. Mu.M FIP/BIP primer pair, 0.2. Mu. M F3/B3 primer pair, 0.8. Mu.M LoopB primer, 1.4 mM dNTP, syto dye, and 0.32U/mu.L Bst DNA polymerase. The methods, lysates, or amplification compositions of the invention are useful for increasing the detection sensitivity and detection rate of nucleic acids in a viscous biological sample in LAMP amplification, enabling detection of low-load nucleic acid samples, and increasing the efficiency of nucleic acid detection in a viscous biological sample.
Advantageous effects
The method and the application thereof can improve the detection efficiency of nucleic acid in a viscous biological sample, reduce the inhibition of polysaccharide components in the sample to amplification reaction by adding a proper amount of SrCl 2 into a lysate, improve the detection sensitivity and the detection rate of nucleic acid in the viscous biological sample in LAMP amplification, and realize the detection of a low-load nucleic acid sample.
Drawings
FIGS. 1A-1B show the effect of SrCl 2 on Bst enzyme activity;
FIG. 2 is a graph showing the effect of SrCl 2 on LAMP amplification reaction;
FIGS. 3A-3C show the effect of SrCl 2 on the LAMP amplification reaction of polysaccharide-containing nucleic acid samples;
FIGS. 4A-4C illustrate LAMP amplification reactions of oral swab samples.
Detailed Description
The technical scheme of the present disclosure is further described below in conjunction with specific embodiments. The following embodiments are merely examples of the present disclosure and do not represent or limit the scope of the present disclosure. The protection scope of the present disclosure is subject to the claims. In the examples below, reagents and consumables were purchased from commercial suppliers, and experimental methods and techniques were used as conventional in the art, unless otherwise specified.
Example 1 SrCl 2 Effect on LAMP amplification reaction
1.1 Effect of SrCl 2 on Bst enzyme activity:
(1) Primer design and screening, namely designing and testing a sequence modified by a fluorescent group and a quenching group of Bst enzyme activity:
5' FAM-Template sequence (SEQ ID NO: 1): CGACGTGTCTGCGGCTCTACCATATCTCCTATGAGCAACGTGTTAGCAGAGCCAAGCCACAACTC
3' BHQ1-Reporter sequence (SEQ ID NO: 2): TGCTCATAGGAGATATGGTAGAGCCGCAGACACGTCG
Cold PRIMER DNA sequence (SEQ ID NO: 3):
GAGTTGTGGCTTGGCTCTGCTAACACG。
(2) Bst enzyme activity test reaction system was prepared according to Table 2:
TABLE 1
(3) After mixing, 60 min was amplified at 65 ℃ in a qPCR instrument, with fluorescent signals collected every 1:1 min.
As a result of the detection, as shown in FIG. 1A, the slope of the fluorescence curve was positively correlated with Bst enzyme activity, which decreased with an increase in SrCl 2 (1 mM-5 mM).
1.2 Effect of SrCl 2 in viscous biological sample lysates on Bst enzyme activity
(1) Primer design screening, refer to step (1) in 1.1.
(2) Oral swab sample preparation the swab was scraped and placed in 400 μl of lysate (Tris, 1mM EDTA, srCl2, 1 w/v% SDS, and 2 w/v% PEG200 at pH 7.0, 10 mM) with SrCl2 concentrations of 0mM, 0.5 mM, 5mM, 50 mM, 100mM SrCl 2, respectively, and room temperature lysed 5min to prepare an oral swab lysed sample. The control was lysate or nucleic-free ddH 2 O.
(3) Bst enzyme activity test reaction system was prepared according to Table 2:
TABLE 2
(4) After mixing, amplifying for 60min at 65 ℃ in a qPCR instrument, and collecting a fluorescent signal for 1 min.
The detection result is shown in FIG. 1B, and the slope of the fluorescence curve is positively correlated with Bst enzyme activity.
1.3 Effect of SrCl 2 on LAMP amplification reaction
(1) Designing and screening primers, namely designing and synthesizing LAMP primers according to the SA-GAPA1 gene sequence.
FIP(SEQ ID NO:4):
GTGAAACGACCTTGCATAGTGTCGTAAACGACTTAACAGATGACG
BIP(SEQ ID NO:5):
GGTGAAGTAGAGGTAGTTGATGGTATTTGCTTGCATCTGGTTC
F3(SEQ ID NO:6):
GAAGGTCTTGAAGTTGTAGC
B3(SEQ ID NO:7):
CATCGATATTTAAGTCTTTCCAAG
LoopB(SEQ ID NO:8):
GTTTCCGCGTAAATGGTAAAGAAGT
(2) LAMP reaction systems were formulated as in table 3:
TABLE 3 Table 3
(3) After mixing, 60 min was amplified at 65 ℃ in a qPCR instrument, with fluorescent signals collected every 1:1 min.
The detection results are shown in FIG. 2, srCl 2 of 1-5 mM has no obvious effect on the LAMP amplification reaction of higher concentration (10 pg/rxn) nucleic acid, but SrCl 2 of 5mM has obvious inhibition effect on the LAMP amplification reaction in the amplification process of low concentration (1 or 0.1 pg/rxn) nucleic acid.
Example 2 SrCl 2 promotion of LAMP amplification reaction of polysaccharide-containing samples
(1) Primer design screening synthetic LAMP primers were designed based on the SA-GAPA1 gene sequence, see step (1) of example 1, 1.3.
(2) LAMP reaction systems were formulated as in table 4:
TABLE 4 Table 4
(3) After mixing, 60 min was amplified at 65 ℃ in a qPCR instrument, with fluorescent signals collected every 1:1 min.
The detection results are shown in FIGS. 3A-3C, and in the samples containing polysaccharide (0.1% glucose sulfate), srCl 2 of 1 and 5mM can effectively improve the amplification efficiency of LAMP reaction and the detection rate of target nucleic acid.
EXAMPLE 3 LAMP amplification reaction of viscous biological samples (oral swabs)
(1) Primer design screening synthetic LAMP primers were designed based on the SA-GAPA1 gene sequence, see step (1) of example 1, 1.3.
(2) Oral swab sample preparation the swab was scraped and placed in 400 μl of lysate (Tris, EDTA, srCl2, SDS, and PEG200 at pH 7.0, 10 mM, 1 mM, 1 w/v% and 2 w/v%), wherein the concentration of SrCl 2 in the lysate was 0 mM, 5 mM, 25 mM, respectively, and lysed at room temperature 5 min to prepare an oral swab lysate sample. A blank swab lysis sample (0 mM SrCl 2) was used as a control, i.e., lysate control.
(3) LAMP reaction systems were formulated as in table 5:
TABLE 5
(4) After mixing, 60 min was amplified at 65 ℃ in a qPCR instrument, with fluorescent signals collected every 1:1 min.
The detection results are shown in fig. 4A-4C, and after the oral swab sample is cracked by using the lysate containing 5mM or 25mM SrCl 2, the amplification efficiency and the target nucleic acid detection rate of the cracked sample in the LAMP detection reaction can be effectively improved.
The above description is merely illustrative of the preferred embodiments of the present invention and should not be taken as limiting the scope of the invention, which is defined by the appended claims.
Claims (6)
1. A method of detecting nucleic acid in a biological sample, comprising:
(1) Releasing nucleic acid in the viscous biological sample by using the lysate to obtain a sample lysate; (2) sampling the lysate to carry out LAMP amplification reaction, and is characterized in that the lysate contains Sr 2+;
the concentration of Sr 2+ in the lysate is 0.5-25mM;
Sr 2+ in the pyrolysis liquid is generated after SrCl 2 is added;
The lysate also comprises a buffer component, a surfactant, a stabilizer and a chelating agent, wherein the buffer component is 5-50mM Tris, the surfactant is 0.5-10w/v% SDS, the stabilizer is 1-10w/v% PEG200, the chelating agent is 0.5-10mM EDTA, and the pH of the lysate is 7.0-9.0.
2. The method of claim 1, wherein the viscous biological sample is selected from the group consisting of sputum, cervical mucus, nasopharyngeal swab, oral swab, genital swab, and lavage.
3. A lysate comprising Sr 2+, the concentration of Sr 2+ being selected from 0.5-25mM, sr 2+ in the lysate being produced by adding SrCl 2;
The lysate also comprises a buffer component, a surfactant, a stabilizer and a chelating agent;
the buffer component is 5-50mM Tris, the surfactant is 0.5-10w/v% SDS, the stabilizer is 1-10w/v% PEG200, the chelating agent is 0.5-10mM EDTA, and the pH of the lysate is 7.0-9.0.
4. A kit for detecting nucleic acid in a biological sample, comprising the lysate of claim 3.
5. The kit of claim 4, further comprising a LAMP amplification system.
6. The kit of claim 5, wherein the LAMP amplification reaction system comprises 1 Xbuffer, mgSO4, dNTPs, bst DNA polymerase, primer set, and fluorescent indicator.
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