CN117327841B - Method for jointly detecting monkey pox virus and chikungunya virus by capillary modified LAMP method - Google Patents
Method for jointly detecting monkey pox virus and chikungunya virus by capillary modified LAMP method Download PDFInfo
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- 238000007397 LAMP assay Methods 0.000 title claims abstract description 37
- 241001502567 Chikungunya virus Species 0.000 title claims abstract description 27
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- 238000000034 method Methods 0.000 title claims abstract description 19
- 238000001514 detection method Methods 0.000 claims abstract description 50
- 238000006243 chemical reaction Methods 0.000 claims description 13
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 6
- 238000006911 enzymatic reaction Methods 0.000 claims description 6
- 239000010902 straw Substances 0.000 claims description 5
- 238000004458 analytical method Methods 0.000 claims description 4
- 239000007793 ph indicator Substances 0.000 claims description 3
- 238000011534 incubation Methods 0.000 claims description 2
- 238000003745 diagnosis Methods 0.000 claims 4
- 201000010099 disease Diseases 0.000 claims 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims 2
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 238000010438 heat treatment Methods 0.000 abstract description 2
- 239000013612 plasmid Substances 0.000 description 15
- 239000000203 mixture Substances 0.000 description 9
- 101800000120 Host translation inhibitor nsp1 Proteins 0.000 description 7
- 101800000512 Non-structural protein 1 Proteins 0.000 description 7
- 239000000443 aerosol Substances 0.000 description 4
- 239000012154 double-distilled water Substances 0.000 description 4
- 238000007689 inspection Methods 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 150000003839 salts Chemical class 0.000 description 4
- 241000700605 Viruses Species 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000001502 gel electrophoresis Methods 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- 238000004383 yellowing Methods 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 239000012880 LB liquid culture medium Substances 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000010241 blood sampling Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- XZTWHWHGBBCSMX-UHFFFAOYSA-J dimagnesium;phosphonato phosphate Chemical compound [Mg+2].[Mg+2].[O-]P([O-])(=O)OP([O-])([O-])=O XZTWHWHGBBCSMX-UHFFFAOYSA-J 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
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- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000013492 plasmid preparation Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
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Abstract
The invention relates to the technical field of LAMP detection, in particular to a method for jointly detecting monkey pox virus and chikungunya virus by a capillary improved LAMP method, which is characterized in that the use of the capillary is more convenient in the joint detection process, the detection result can be directly observed, the detection by using the LAMP method needs to be heated, the occurrence of a pollution false positive result caused by heating is improved by using the capillary, and the sensitivity and the authenticity of the detection method are improved; and the LAMP method is further optimized, so that the monkey pox virus and the chikungunya virus can be detected in a combined way, the detection cost is reduced, and the detection efficiency is improved.
Description
Technical Field
The invention relates to the technical field of LAMP detection, in particular to a method for jointly detecting monkey pox virus and chikungunya virus by a capillary modified LAMP method.
Background
Currently there are no approved related vaccines and specific drugs for monkey pox virus and chikungunya virus, so inspection and quarantine work for these two viruses is a key to controlling cross-border input.
Among the detection techniques commonly used at present, one is a conventional PCR method, and one is a loop-mediated isothermal amplification (LAMP) technique. The LAMP method is a brand-new nucleic acid amplification method, and is characterized in that four primers are designed for six regions on a target gene, amplification reaction is carried out by using strand displacement type DNA polymerase under the constant temperature condition, 10 9-1010 times of amplification can be realized within 15-60 minutes, a large amount of amplification products, namely white precipitation of magnesium pyrophosphate, can be generated by the reaction, and whether the target gene exists can be judged by observing whether the white precipitation is generated or not through naked eyes. The LAMP method has the characteristics of simplicity, rapidness and strong specificity, can compare favorably with or even be superior to the PCR technology in indexes such as sensitivity, specificity, detection range and the like, does not need the processes of thermal denaturation, temperature circulation, electrophoresis, ultraviolet observation and the like of a template, does not depend on any special instrument and equipment to realize on-site high-flux rapid detection, has detection cost far lower than fluorescent quantitative PCR, and is a method suitable for on-site and basic-layer rapid detection.
The LAMP method has the advantages of high specificity, high sensitivity and very simple operation, and the reaction conditions are easy to control, and the LAMP method is simple in equipment, is very suitable for the inspection and quarantine of overseas input cases, and is widely applied. In the experimental process, the experimenters find that the final detection result is affected by different vectors of the primer and the substance to be detected, so that the detection of the monkey pox virus and the chikungunya virus by the LAMP method is required, and the LAMP method is further required to be optimized.
In the experimental process, experimental staff also find that if the common EP tube in the detection experiment is used for the detection experiment, false positive results often appear, and the analysis of experimental data deduces that the final data result is unstable possibly due to aerosol pollution, and meanwhile, the sample acquisition dosage is large, so that cost waste is caused. Therefore, the experimenter considers the mode of using the capillary tube commonly used for blood sampling to replace the EP tube, and the capillary tube is made of glass material, so that aerosol pollution can be avoided, and the cost is reduced, so that in order to further improve the detection efficiency and the data reliability, an improved LAMP detection method for the capillary tube is urgently needed, and the gap of inspection and quarantine of the monkey pox virus and the chikungunya virus is made up.
Disclosure of Invention
The invention aims to provide a capillary tube improved LAMP detection method;
another object of the invention is a capillary modified LAMP assay for the combined detection of monkey pox virus and chikungunya virus.
The technical scheme adopted for realizing the purpose of the invention is as follows:
1. and (3) configuring a reaction system:
The LAMP method of Bst4.0 red Huang Bianse is adopted, and the standard reaction system of Bst4.0 red yellowing is 9 mu L with the red pH indicator dye, and is as follows: 2.5xBst4.0 LS MasterMix 4. Mu.L, 10xLAMP Primer Mix 1. Mu.L, 1mM NaOH was added to the 10 Xprimer to restore the primer to neutral pH (pH=7 to 8.5), the primer was diluted to 10. Mu.M with ddH 2 O, and the volume was made up to 9. Mu.L with ddH 2 O.
Wherein, the primer is designed by LAMP primer of MPXV F13 and CHIKV nsp1 by PrimerExplorerV software, and the primer synthesis is carried out by Beijing qingke organism, the primer information is as follows:
Meter 1.Monkeypox virus F13 LAMP primer
Meter 2.Chikungunya virus nsp1 LAMP primer
Specifically, the monkey pox virus Bst4.0Low SALT MASTER Mix LAMP enzymatic reaction-9. Mu.L reaction system comprises ：2.5xBst4.0 LS Master Mix 4μL、F13-F3 0.1μL、F13-R3 0.1μL、F13-FIP 0.4μL、F13-BIP 0.4μL、ddH2O 4μL;
Specifically, the chikungunya virus Bst4.0 Low SALT MASTER Mix LAMP enzymatic reaction-10. Mu.L reaction system comprises ：2.5x Bst4.0 LS Master Mix 4μL、nsp1-F3 0.1μL、nsp1-R3 0.1μL、nsp1-FIP 0.4μL、nsp1-BIP 0.4μL、ddH2O 4μL.
2. And (3) detection:
taking capillary straws with different amounts Cheng Kedu to suck 9 mu L of solution in the enzyme reaction system which is mixed in advance, then sucking 1 mu L of corresponding sample to be tested, and placing the sample in a 65 ℃ incubator for 30min.
3. Analysis of results:
After reacting for 30min, taking out the capillary, and directly observing the color change of the indicator by naked eyes: the detection result is regarded as positive when the color of the red color is changed into yellow; negative if no change occurs.
Compared with the prior art, the invention has the beneficial effects that:
the LAMP detection method has the advantages that the established experimental data are accumulated, the data obtained by the detection method are proved to be true and effective, the method can replace the traditional PCR technology, and becomes an important molecular biology research tool.
2. The capillary tube is used for replacing an EP tube, and the LAMP detection method is optimized to jointly detect the monkey pox virus and the chikungunya virus, so that the problem of aerosol pollution generated in a high-temperature environment is solved, and the phenomenon that substances in the capillary tube are evaporated is avoided in the high-temperature environment, so that the detection result data is more stable and the accuracy is higher; when an EP tube is used for LAMP detection, a detection system of 25 mu L is used, after a capillary tube is used, only 10 mu L of detection system is needed, and the sampling amount is reduced by only sucking 1-1.5 mu L of sample each time, so that the accuracy of a detection result is not affected, the sensitivity is high, and 6 copies can be detected even lower; and the capillary tube can automatically suck samples, so that the contact time of an experimenter in detecting samples is reduced, and the capillary tube has the characteristics of convenient observation and flexible replacement, so that the optimized LAMP detection method is used for jointly detecting the monkey pox virus and the chikungunya virus.
3. Although the detection method is still in a laboratory stage at present, and the scheme only carries out the detection of plasmid samples, reasonable expectation can be made, the method can be applied to the actual detection work of the monkey pox virus and chikungunya virus, and the blood sample can be directly sucked by a capillary tube which has sucked the enzymatic reaction system solution by utilizing the method for detection, so that the detection time is shortened, and the detection efficiency is improved; is favorable for inspection and quarantine work and reduces the exposure risk of the monkey pox virus and the chikungunya virus.
Drawings
FIG. 1 shows the results of detection of monkey pox virus by the capillary modified LAMP method in example 3 of the present invention;
FIG. 2 shows the results of detection of chikungunya virus by capillary modified LAMP in example 3 of the present invention;
FIG. 3 shows the detection results of monkey poxvirus by gel electrophoresis;
FIG. 4 shows the results of detection of chikungunya virus by gel electrophoresis.
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other.
Example 1 preparation of samples to be tested
Since the blood sample was difficult to obtain, the present example used a viral plasmid as an experimental sample, and the viral plasmid preparation process was as follows:
The monkey pox virus F13 (sequence information MPXV USA2022MA001, source NCBI GenBank: ON 563414.3) (1119 bp) and chikungunya virus nsp1 (1605 bp) (sequence information chiKV SK001/12, source NCBI GenBank: ON 262792.1) are respectively used as target gene indicators for detecting viruses, and are respectively used as the basis for detecting MPXV and chiKV, and the sequences are synthesized and constructed into PLVX-EGFP-C1 (8786 bp) and 3XFlag-CMV-14 (6310 bp) vectors by Beijing engine biotechnology Co.
Single bacterial colony is selected by streaking DH5 alpha strain containing CHIKV nsp1 and MPXV F13 full-length gene plasmid constructed by the Optimago company through LB solid culture medium flat plate, plasmid is extracted by a plasmid extracting kit in the radix angelicae sinensis after shaking and amplifying by 12-16 h LB liquid culture medium at 37 ℃, and plasmid concentration is measured by a spectrophotometer nanodrop8000 respectively: 578 ng/. Mu.L and 983 ng/. Mu.L (copy number calculation formula: (6.02X10 23) × (plasmid concentration ng/. Mu.L X10 -9)/(constructed plasmid DNA LENGTH X660) =copies/. Mu.L.).
The concentration of the extracted plasmid in the MPXV F13 stock solution is as follows: (6.02X10 23)×(983ng/μL×10-9)/(9905bp×660)≈1.0×1011 copies/. Mu.L, ten-fold gradient dilution to Standard concentration) ：1×106copies/μL,1×105copies/μL,1×104copies/μL,1×103copies/μL,1×102copies/μL,10copies/μL.
The concentration of plasmids extracted from the CHIKV nsp1 stock solution is as follows: (6.02X10 23)×(578ng/μL×10-9)/(7915bp×660)＝6.5×1010 copies/. Mu.L, ten-fold gradient dilution to Standard concentration) ：6×105copies/μL,6×104copies/μL,6×103copies/μL,6×102copies/μL,6×101copies/μL,6copies/μL.
Example 2 Combined detection of Simian poxvirus and chikungunya Virus
The LAMP method of Bst4.0 red Huang Bianse is adopted, and the LAMP enzymatic reaction of the monkey pox virus Bst4.0 Low SALT MASTER Mix is matched with a red pH indicator dye, wherein a reaction system of 9 mu L comprises ：2.5x Bst4.0 LS Master Mix 4μL、F13-F3 0.1μL、F13-R3 0.1μL、F13-FIP 0.4μL、F13-BIP 0.4μL、ddH2O 4μL; chikungunya virus Bst4.0 Low SALT MASTER Mix and a reaction system of 9 mu L comprises ：2.5x Bst4.0 LS Master Mix 4μL、nsp1-F30.1μL、nsp1-R3 0.1μL、nsp1-FIP 0.4μL、nsp1-BIP 0.4μL、ddH2O 4μL.
Taking 12 capillary straws with different amounts Cheng Kedu, respectively sucking 9 mu L of each solution in the two enzymatic reaction systems which are mixed in advance, using 6 capillary straws for each system, respectively sucking 1 mu L of each 6 gradient virus plasmid sample to be detected of the monkey pox virus and the chikungunya virus prepared in the example 1, placing the 12 capillary straws in a 65 ℃ incubator for incubation for 30min, and taking out the experimental capillary to observe the color change.
Example 3 analysis of experimental results
MPXV F13 plasmid concentration 1×106copies/μL,1×105copies/μL,1×104copies/μL,1×103copies/μL,1×102copies/μL,10copies/μL and chiKV nsp1 plasmid concentration 6×105copies/μL,6×104copies/μL,6×103copies/μL,6×102copies/μL,6×101copies/μL,6copies/μL were detected, respectively, the observation results are shown in FIG. 1, and the electrophoresis gel verification is performed, and the results are shown in FIGS. 2 and 3.
As can be seen from FIG. 1, when MPXV F13 plasmid was detected, even if the standard solution diluted to 10 copies/. Mu.L was detected by the LAMP method using a capillary, the change of the color of the indicator was visually observed by naked eyes, and when CHIKV nsp1 plasmid was detected, the yellowing of the indicator in the capillary was still observed when diluted to 6X 10 1 copies/. Mu.L, indicating that MPXV and CHIKV could be detected by the improved LAMP method using a capillary, and the sensitivity was high, and the detection requirement could be satisfied even if the sampling amount was only 10 copies/. Mu.L, and the accuracy of the experimental result was verified by electrophoresis gel, wherein no false positive data was present, and the influence of the generation of aerosol phenomenon after heating on the result was solved.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (4)
1. A method for jointly detecting monkey pox virus and chikungunya virus by a capillary modified LAMP method for the purpose of non-disease diagnosis is characterized by comprising the following steps:
(1) And (3) configuring a reaction system: preparing a 9 mu L reaction system, wherein the reaction system is a LAMP method system of Bst4.0 red Huang Bianse and is matched with a red pH indicator dye; the synthetic primers in the system comprise a monkey pox virus primer and a chikungunya virus primer, wherein the amino acid sequence of the monkey pox virus detection primer is as follows:
F13-F3 5‘-GATGTTATGTAGGAAACGCG-3’
F13-R3 5‘-AACACTCCACCTATAGGATTC-3’
F13-FIP 5‘-GCCAGCGGAGGATAATCAGAATATATTTACTGGAGGATCTATACATACG-3’
F13-BIP 5‘-ATAGCGCAAAAAATTCATGGTTGAAATATGATACGCAGTGCTAAC-3’；
the amino acid sequence of the chikungunya virus detection primer is as follows:
nsp1-F3 5‘-CAACCATCCTGGATATCGGC-3’
nsp1-R3 5‘-GTTGGCGTCTCCTTGTCTG-3’
nsp1-FIP 5‘-GGATCTTCCGCACTGCGCATCCAGCAAGGAGGATGATGTCG-3’
nsp1-BIP 5‘-CCGCAGGAAAAGTCCTGGACACACGGCCATTACTGCTTGT-3’；
(2) And (3) detection: taking capillary straws with different amounts Cheng Kedu to absorb the solution in the enzymatic reaction system which is mixed in advance, then absorbing the corresponding sample to be tested, and placing the sample in a 65 ℃ incubator for incubation for 30min;
(3) Analysis of results: the naked eyes directly observe that the color of the indicator changes from red to yellow, and the detection result is regarded as positive; negative if no change occurs.
2. The method for combined detection of monkey pox virus and chikungunya virus by capillary modified LAMP for non-disease diagnosis as claimed in claim 1, wherein in the step (1), the reaction system is as follows: 2.5xBst4.0 LS MasterMix 4. Mu.L, 10xLAMP Primer Mix 1. Mu.L, and the volume was made up to 9. Mu.L with ddH 2 O.
3. The method for combined detection of monkey pox virus and chikungunya virus by capillary modified LAMP for non-disease diagnosis according to claim 2, wherein the 10X primer is diluted to 10. Mu.M with ddH 2 O by adding NaOH to adjust the pH of the primer to 7 to 8.5 at a final concentration of 1 mM.
4. The method for jointly detecting a monkey pox virus and a chikungunya virus by a capillary modified LAMP method for the purpose of non-disease diagnosis according to claim 1, wherein the amount of the sample of the enzymatic reaction system solution in the step (2) is 9. Mu.L and the amount of the sample to be detected is 1. Mu.L.
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| CN109996888A (en) * | 2016-09-23 | 2019-07-09 | 阿尔韦奥科技公司 | For testing and analyzing the method and composition of object |
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|---|---|---|---|---|
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| CN204958934U (en) * | 2015-08-17 | 2016-01-13 | 广州市第一人民医院 | A mancarried device that is used for capillary isothermal to amplify and detect |
| CN109996888A (en) * | 2016-09-23 | 2019-07-09 | 阿尔韦奥科技公司 | For testing and analyzing the method and composition of object |
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| Title |
|---|
| Development of a Novel Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Monkeypox Virus Infections;Chao Yu 等;VIRUSES;20221231;第15卷(第84期) * |
| Simpler, Faster, and Sensitive Zika Virus Assay Using Smartphone Detection of Loop-mediated Isothermal Amplification on Paper Microfluidic Chips;Kattika Kaarj 等;Scientific ReportS;20180820(第8期);文献号：12438 * |
| 基孔肯雅病毒LAMP 检测方法的建立及其应用研究;李小波 等;中国卫生检验杂志;20120331;第22卷(第3期);506-508 * |
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