CN115747310A - Fluorescent probe based on RPA-LFD detection and preparation method and application thereof - Google Patents
Fluorescent probe based on RPA-LFD detection and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a fluorescent probe based on RPA-LFD detection and a preparation method and application thereof, belonging to the technical field of detection. In order to provide a fluorescent probe for improving the sensitivity and accuracy of an RPA-LFD detection kit. The invention provides a fluorescent probe based on RPA-LFD detection, which has the following sequence: [ FAM ] -CTTTCTGCTTCTGTAATTGATGCCTTGTTTT- [ THF ] -CATGATACTTGTTTG- [ C3Spacer ]. The method is applied to the detection of the parasitic diseases.
Description
Technical Field
The invention belongs to the technical field of detection, and particularly relates to a fluorescent probe based on RPA-LFD detection, and a preparation method and application thereof.
Background
Clonorchis sinensis is an important food-borne parasitic zoonosis which is parasitic in the hepatic bile ducts and gall bladder of human and various mammals and causes clonorchis sinensis. In 2009 WHO classified clonorchis sinensis as a class I biological carcinogenic factor, biliary epithelial carcinoma was caused by patients with biliary tract obstruction, cholangitis and cirrhosis. More than 2 hundred million people in the world have the risk of infecting clonorchis sinensis, 18 provinces in China have reports of clonorchiasis sinensis, the number of infected people reaches as much as 600 ten thousands, and the prevention and control of clonorchiasis sinensis cannot be ignored.
The diagnosis method of clonorchiasis sinensis includes etiology, imaging, molecular biology, immunology, etc., but the detection of the ovum in feces is still the main standard for diagnosing the disease. The methods of etiologic diagnosis have certain advantages, which also have drawbacks: for example, because the eggs of the clonorchis sinensis and the eggs of the epididymis are relatively similar, the requirements on the experience and the specialty of operators are higher; false or missed detection is prone to occur during mild infections. Enzyme-linked immunosorbent assays (ELISA), immunochromatographic assays (ICTs) and time-resolved fluoroimmunoassay (TRFIA) in the immunological methods have high sensitivity and are useful for epidemiological studies and large-scale disease monitoring, but they are prone to high cross-reactivity and false positive rate and difficult to distinguish between current and previous infections. Molecular biological methods have high sensitivity and specificity, and various PCR-based diagnostic methods have been developed to detect the DNA of the sample Clonorchis sinensis, such as conventional PCR, real-time PCR, nested PCR, multiplex PCR, etc., although PCR-based detection has high sensitivity and can detect a low number of parasite genomes, the need for specific tools (such as PCR instruments, etc.) limits its application in resource-limited environments.
Recombinase polymerase amplification combined with lateral flow chromatography test paper (RPA-LFD) is an isothermal DNA amplification technology, and mainly depends on recombinase, single-strand binding protein (SSB) and DNA polymerase to realize amplification of a target gene at a constant temperature, and the method can finish amplification of the target gene within 20 minutes. In addition, the RPA amplification product can be further combined with detection equipment such as a test strip, so that the detection result can be easily read, the endpoint analysis is more flexible, the accurate, rapid and visual detection target is realized, and the problem that the field detection cannot be performed in part of basic detection laboratories due to the lagging equipment is solved.
Disclosure of Invention
The invention aims to provide a fluorescent probe for improving the sensitivity and accuracy of an RPA-LFD detection kit.
The invention provides a fluorescent probe based on RPA-LFD detection, which has the following sequence: [ fluorophore ] -CTTTCTGCTTCTGTAATTGATGCCTTGTTT- [ THF ] -CATGATACTTGTTTG- [ quencher ].
Further defined, the fluorescent group is FAM and the quenching group is C3 Spacer.
Further defined, the formula of FAM is C 27 H 18 N 2 O 8 (ii) a C3 The molecular formula of Spacer is C 33 H 43 N2O5P; the molecular formula of THF is C4H8O.
The invention provides a preparation method of the fluorescent probe, which is characterized by comprising the following steps: the fluorescent probe is obtained by modifying a 5' end in a CTTTCTGCTTCTGTAATTGATGCCTTGTTTTCATGATATATACTTGGTTTG probe sequence by a FAM fluorescent group, inserting a THF group at a position 30bp away from the 5' end, and modifying a 3' end by a C3-Spacer group.
The invention provides the application of the fluorescent probe or the preparation method in preparing a kit for detecting clonorchis sinensis.
The invention provides a kit for detecting clonorchis sinensis, which comprises the fluorescent probe, an upstream primer, a downstream primer coupled with biotin at the 5' end and a starter.
Further defined, the sequence of the downstream primer coupled with biotin at the 5' end is as follows: biotin-TACAGAATAACAACCGTCCTAAACGACCTA.
Further defined, biotin has the molecular formula C10H18N4O2S.
Further defined, the starter composition is 280mM magnesium acetate.
Further defined, the reaction system of the kit is 50 μ L: mu.L reaction buffer, 10. Mu.M 2.1. Mu.L upstream primer, 10. Mu.M 2.1. Mu.L downstream primer, 10. Mu.M 0.6. Mu.L probe, 16.2. Mu.L ddH2O, 1. Mu.L clonorchis sinensis DNA,280 mmol/L3. Mu.L promoter.
Has the advantages that: 1. short time and high efficiency: the RPA amplification process only needs 20min, and compared with the traditional PCR, the reaction time is greatly shortened;
2. the amplification temperature is constant: the RPA can complete the amplification of the target gene at the constant temperature of 37 ℃, the whole process does not need high-temperature denaturation and low-temperature annealing steps, expensive instruments (such as a PCR instrument and the like) are not needed, and the problem that part of basic detection points can not be detected on site due to laggard equipment is solved.
3. The operation is simple and convenient: the enzyme and the reagent required by amplification are placed in an independent reaction tube in a powder form and can be placed at normal temperature, the reaction can be started only by adding corresponding buffer solution, primers, probes, templates and a starter during amplification, the requirement on operators is low, and the kit is suitable for field diagnosis of a basic detection point;
4. the specificity is strong: the kit is added with the probe, can be used for accurately diagnosing the clonorchis sinensis and has no cross reaction with other parasites;
5. the sensitivity is high: the primer and the probe of clonorchis sinensis RPA designed by the invention can realize the nucleic acid detection limit of 100fg at the temperature of 37 ℃ under the condition of 20min amplification.
Three groups of [ FAM ] -CTTTCTGCTTCTGTAATTGATGCCTTGTTTT- [ THF ] -CATGATACTTGGTTTG- [ C3Spacer ] are required to be added, wherein THF cannot be replaced, a tetrahydrofuran modified site is introduced into a probe, and the tetrahydrofuran modified nucleotide can realize DNA chain extension with almost 100% efficiency under the action of DNA polymerase; when the probe and a target sequence structure form double-stranded DNA, if the Nfo enzyme meets a tetrahydrofuran modified site in the patrol process, the damage of the DNA is considered to be repaired, so that the site on the probe is sheared, and a fluorescent group is released; the cleaved probe has a free 3' -OH group, and thus can be used as a primer to continue synthesis of a new template. FAM and C3Spacer are fluorescent and quenching groups, and can be replaced by other similar functional groups.
Drawings
FIG. 1 is a schematic diagram illustrating the determination of the RPA-LFD detection result of the present invention. Wherein T is a detection line; c is a quality control line; a is a schematic diagram of invalid results; b is a negative result diagram; c is a positive result diagram;
FIG. 2 shows the specific analysis of RPA-LFD according to the present invention. Wherein T is a detection line; c is a quality control line; a is a negative control; b is the oriental subthreshold; c is trichina; d is anisakis; e is clonorchis sinensis;
FIG. 3 is an RPA-LFD sensitivity assay of the present invention;
FIG. 4 shows the sensitivity analysis of general PCR, where M is DNA Marker, and the concentrations of 1-8 template DNAs are 100ng, 10ng, 1ng, 100pg, 10pg, 1pg, 100fg, and 10fg in this order; 9 is a negative control.
Detailed Description
Example 1 preparation of fluorescent probes
[FAM]-CTTTCTGCTTCTGTAATTGATGCCTTGTTT-[THF]-CATGATACTTGGTTTG-[C3 Spacer]
The preparation method comprises the following steps: the probe is synthesized by the company of Biotechnology engineering (Shanghai), inc., and comprises the following components: probe 5'- (FAM) label, with a tetrahydrofuran residue (THF) inserted between 30 and 31, and a C3Spacer inserted at the 3' end.
FAM molecular formula: c 27 H 18 N 2 O 8 The chemical formula is as follows:
c3 The Spacer molecular formula: c 33 H 43 N 2 O 5 P is a chemical formula:
THF formula: c 4 H 8 The chemical formula of O:
example 2 clonorchis sinensis COX1 gene primers
The primer and the probe are designed aiming at the clonorchis sinensis COX1 gene sequence (GenBank: MF 287785). And (3) comparing and analyzing the sequences by MEGA X software, selecting the conserved region as a Primer candidate region, and setting selection conditions by using Primer 5 software. Setting primer parameters: length, 30-35bp; GC content, 30-65%; annealing temperature, 52-58 ℃. The primers were synthesized by Biotechnology engineering (Shanghai) Inc., as shown in Table 1.
Table 1. The sequence of the primer and probe of clonorchis sinensis RPA designed by the invention:
biotin was given to the company for synthetic coupling, biotin molecular formula: c 10 H 18 N 4 O 2 The chemical formula of S is as follows:
example 3 establishment of RPA-LFD detection method
1. Clonorchis sinensis genome extraction
The clonorchis sinensis strain used in the invention is stored in the laboratory, clonorchis sinensis adult genome DNA is extracted by utilizing a TIANMP Genomic DNAkit genome DNA kit of TIANGEN company, and feces sample DNA is extracted by utilizing a TIANGEN feces DNA rapid extraction kit specification and then dissolved in TE and stored at-20 ℃.
RPA reaction System and conditions
The reagentThe reaction system of the cartridge was 50 μ L: 25. Mu.L of reaction buffer, 2.1. Mu.L of forward primer (RPA-F, 10. Mu.M), 2.1. Mu.L of reverse primer (Biotin-TACAGAATAACAACCGTCCTAAACGACCTA, 10. Mu.M), 0.6. Mu.L of probe ([ FAM)]-CTTTCTGCTTCTGTAATTGATGCCTTGTTT-[THF]-CATGATACTTGGTTTG-[C3 Spacer]，10μM)，16.2μL ddH 2 O, 1. Mu.L of clonorchis sinensis genomic DNA, 3. Mu.L of promoter (280 mM magnesium acetate). After 47. Mu.L of the reaction reagent except the cooling promoter was premixed, the premixed reagent was added to a separate reaction tube containing RPA lyophilized powder (RPA reaction kit TwistAmptM Basic kit), and after sufficient dissolution, 3. Mu.L of the promoter was added. The reaction temperature for RPA amplification is 37 deg.C, and the reaction time is 20min.
Detection of RPA amplification product
The lateral flow chromatography test strip used in the invention comprises a sample pad, a chromatography pad and a water absorption pad in sequence on a bottom plate along the chromatography direction during sample detection. The prepared streptavidin and the anti-FAM antibody are respectively sprayed on a chromatographic membrane to form a detection line and a quality control line, dried at 37 ℃ for 2-3h, sealed and stored. Diluting the amplification product by PBST buffer solution for 20 times, sucking 40 mu L of the diluted amplification product, dripping the diluted amplification product on a sample pad, and judging the result within 5-10min, wherein the judgment result is shown in figure 1. When the quality control line and the detection line are simultaneously developed, the test line is judged to be positive, and the sample contains clonorchis sinensis genome DNA; when the quality control line color development detection line does not develop color, the detection line is judged to be negative, and the sample does not contain clonorchis sinensis genome DNA; if the quality control line does not develop color, the experiment is judged to be false, and the test result is invalid.
And (3) specific detection: separately using clonorchis sinensis, epididymis orientalis, trichina and heterodera sinensis genome DNA as templates, adding reactants according to the system to perform RPA detection, and performing constant temperature amplification at 37 ℃ for 20min. After the amplification is finished, the amplification product is diluted by 20 times by PBST, 40 mu L of the amplification product is absorbed and dripped into a sample pad, and the result is judged within 5-10 min. The results are shown in fig. 2, only clonorchis sinensis genome DNA can be well amplified, the test strip is positive, and the others are negative, which shows that the method has certain conservation and strong specificity for detecting clonorchis sinensis.
And (3) sensitivity test: the clonorchis sinensis genome DNA is diluted in gradient of 10 times in 100ng-10fg and used as the template for RPA reaction to amplify at 37 deg.c for 20min. After the amplification is finished, the amplification product is diluted by 20 times by PBST, 40 mu L of the diluted amplification product is sucked and dripped on a sample pad, and the result is judged within 5-10 min. As shown in FIG. 3, the value of the test strip positive signal gradually decreases with the decrease of the template concentration, and the positive signal on the detection line can still be seen at the genomic DNA concentration of 100fg, and no positive signal is seen on the detection line at the concentration of 10fg, indicating that the minimum detection limit of the nucleic acid of the invention is 100fg. The detection limit of the common PCR is 100pg, the sensitivity of the kit used by the invention is 1000 times higher than that of the common PCR, and the result is shown in figure 4.
Example 4 clinical sample testing
The stool microscopic examination detection is used for detecting the gold standard of clonorchis sinensis, the stool microscopic examination result is used as a control, the accuracy of RPA-LFD detection can be demonstrated, the rapid detection can be carried out by using the RPA-LFD method under the condition of the same accurate detection, 62 human stool samples from Clouds Fugu city of Jilin province in clonorchis sinensis epidemic area are simultaneously detected by using the RPA-LFD and the stool microscopic examination respectively, the results show that 12 human stool samples in the RPA-LFD and the stool microscopic examination detection samples are positive, the results of the two methods are consistent, and the results are shown in Table 2.
Table 2. Clinical sample testing:
the invention aims at the gene sequence of the mitochondrial cytochrome C oxidase I subunit (COX 1) of the clonorchis sinensis, selects specific regions in interspecies conservation, and screens a set of primer and probe combination which has strong specificity and high sensitivity and can quickly and effectively detect the clonorchis sinensis COX1 gene by designing a plurality of RPA primers and probes, thereby establishing the RPA-LFD detection method. The invention provides an effective technical means for the rapid visual detection of clonorchis sinensis, and overcomes the problem that the field detection cannot be carried out in part of basic level detection laboratories due to the lagging equipment. The primer and the probe designed by the invention have strong specificity, can be used for accurately diagnosing the clonorchis sinensis, and have no cross reaction with other parasites; the sensitivity is high, and the lowest nucleic acid detection limit is 100fg. The invention further verifies the detection accuracy of the kit by utilizing the collected human excrement sample (n = 62), and test data shows that: the results of the gold standard method of the stool microscopic examination are consistent with the results of the RPA-LFD detection method of the invention.
The above examples only express several embodiments of the present invention, but are not to be construed as limiting the scope of the invention. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the spirit of the invention, and these changes and modifications are all within the scope of the invention.
Claims (10)
1. A fluorescent probe based on RPA-LFD detection, which is characterized in that the sequence of the fluorescent probe is as follows: [ fluorophore ] -CTTTCTGCTTCTGTAATTGATGCCTTGTTTT- [ THF ] -CATGATACTTGGTTTG- [ quencher ].
2. The fluorescent probe of claim 1, wherein the fluorophore is FAM and the quencher is C3 Spacer.
3. The fluorescent probe of claim 2, wherein FAM has the formula C 27 H 18 N 2 O 8 (ii) a C3 The molecular formula of Spacer is C 33 H 43 N 2 O 5 P; THF has the formula C 4 H 8 O。
4. The method for preparing a fluorescent probe according to any one of claims 1 to 3, characterized in that the method comprises: the fluorescent probe is obtained by modifying a 5' end in a CTTTCTGCTTCTGTAATTGATGCCTTGTCATGATACTTGGTTTG probe sequence by a FAM fluorescent group, inserting a THF group at a position 30bp away from the 5' end, and modifying a 3' end by a C3-Spacer group.
5. Use of the fluorescent probe according to any one of claims 1 to 3 or the preparation method according to claim 4 for preparing a kit for detecting clonorchis sinensis.
6. A kit for detecting clonorchis sinensis, which comprises the fluorescent probe of claim 1 or 2, an upstream primer, a downstream primer coupled with biotin at the 5' end, and a promoter.
7. The kit according to claim 6, wherein the sequence of the downstream primer coupled with biotin at the 5' end is as follows: biotin-TACAGAATAACAACCGTCCTAAACGACCTA.
8. The kit of claim 7, wherein Biotin has the formula C 10 H 18 N 4 O 2 S。
9. The kit of claim 6, wherein the promoter comprises magnesium acetate.
10. The kit according to claim 6, wherein the reaction system of the kit is 50 μ L:25 μ L reaction buffer, 10 μ M2.1 μ L forward primer, 10 μ M2.1 μ L reverse primer, 10 μ M0.6 μ L probe, 16.2 μ L ddH 2 O, 1. Mu.L of clonorchis sinensis DNA,280mmol/L of 3. Mu.L of promoter.
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