CN108070638B - Recombinase polymerase isothermal amplification method for detecting orientia tsutsutsugamushi, special primer and probe thereof and application - Google Patents
Recombinase polymerase isothermal amplification method for detecting orientia tsutsutsugamushi, special primer and probe thereof and application Download PDFInfo
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Abstract
The present invention provides an RPA detection method for detecting orientia tsutsugamushi, its special primer, probe and its use in detection of orientia tsutsugamushi. The detection method, the special primer and the probe are designed based on the conserved sequence of 56kDa outer membrane protein gene of Orientia tsutsutsugamushi and have oligonucleotide sequences shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4. The invention applies the novel constant temperature amplification technology RPA to the detection of Oriental tsutsugamushi for the first time, the method simulates the enzyme reaction process of DNA replication in vivo, and the specific enzyme and protein combination including recombinase, single-chain binding protein and DNA polymerase is relied on to amplify the DNA template, the amplification of specific nucleic acid sequence can be realized at the constant temperature of 25-43 ℃, and the amplification product can realize visual discrimination through a lateral chromatography test paper. The method has the advantages of high sensitivity, high specificity, low requirement on hardware equipment, short reaction time, no need of complex treatment on samples, suitability for field detection and the like, and is suitable for popularization and application.
Description
Technical Field
The invention belongs to the technical field of biology, relates to the molecular biology of tsutsugamushi disease eastern square and relates to a method for detecting tsutsugamushi disease eastern square and application thereof, in particular to a method for rapidly detecting tsutsugamushi disease eastern square by utilizing recombinase polymerase constant temperature amplification technology (RPA technology), a special primer and a probe thereof and application thereof.
Background
Orientia tsutsutsugamushi (Ot), originally known as Rickettsia tsutsugamushi, belongs to the genus Rickettsia of the family Rickettsia of the order Rickettsia in the class Proteobacteria. The causative agent of tsutsugamushi disease is a strictly intracellular parasitic prokaryotic cell type microorganism. Oriental tsutsugamushi disease can cause tsutsugamushi disease in human, and is transmitted to people by the bite of larvae of tsutsugamushi, with rats as the main source of infection. Mainly prevails in southeast and southwest areas of China, the incidence rate of tsutsugamushi disease rises in recent years, and the natural epidemic situation tends to expand.
Tsutsugamushi disease is characterized by sudden high fever, eschar or ulcer, lymphadenectasis and rash in clinic, some complications such as pneumonia and meningitis are accompanied, and severe cases can cause multi-organ failure and even death. Because the clinical symptoms of the disease are similar to those of other febrile diseases and the disease is insensitive to conventional penicillin and other antibiotics, and a specific detection method aiming at the disease is not established in hospitals at present, misdiagnosis is easily caused clinically, and delayed treatment and even death are caused. At present, detection methods for orientia tsutsugamushi mainly include nonspecific Giemsa staining, specific immunofluorescence antibody staining, enzyme-linked immunosorbent assay, fluorescent quantitative PCR method, and the like. However, these methods have disadvantages of high cost, requiring specific equipment, time consuming and requiring complicated sample handling, resulting in a severely limited range of their practical applications. The establishment of a simple, rapid and field-applicable detection method of orientia tsutsugamushi is of great significance.
In recent years, isothermal nucleic acid Amplification technology has been rapidly developed, wherein Recombinase Polymerase isothermal Amplification (RPA) developed by british TwistDx Inc is known as a nucleic acid detection technology capable of replacing PCR, which is based on Recombinase Polymerase-mediated Amplification principle, simulates an enzyme reaction process of in vivo DNA replication, amplifies a DNA template by depending on specific enzyme and protein combination (Recombinase, single-strand binding protein and DNA Polymerase), can realize Amplification of a specific nucleic acid sequence at a constant temperature of 25-43 ℃, and an Amplification product can realize visual discrimination by a lateral chromatography test strip. The technology has low requirement on hardware equipment and short reaction time, does not need to carry out complex treatment on samples, and is particularly suitable for the fields of in-vitro diagnosis, food safety, biological safety and the like.
Disclosure of Invention
The technical problem to be solved by the present invention is to provide a set of specific primers and probes for rapid detection of orientia tsutsugamushi and a detection method capable of rapidly, simply and specifically detecting orientia tsutsugamushi.
It is therefore a first object of the present invention to provide primers for detecting orientia tsutsugamushi, including two forward primers and two reverse primers. The primer is designed according to the conserved gene 56kDa outer membrane protein gene (GenBank: KM 115577) of Orientia tsutsutsugamushi, which contains a nucleotide fragment of 730 bases and has the nucleotide sequence shown in SEQ ID NO. 1.
Preferably, the length of the primer is 30bp or more.
Preferably, the primers include a forward primer OtF299 and a reverse primer OtR583, two of which are located between nucleotides 299 to 328 and 554 to 583 of the 56kDa outer membrane protein gene (GenBank: KM 115577), respectively, and have oligonucleotide sequences as shown in SEQ ID NO.2 and SEQ ID NO.3, wherein the reverse primer is labeled with Biotin (Biotin) at the 5' end. The double-stranded DNA obtained after amplification of the forward primer and the reverse primer is labeled with biotin.
The special primer for RPA of Orientia tsutsugamushi was designed based on the sequence of 56kDa outer membrane protein gene of Orientia tsutsutsugamushi (GenBank: KM 115577), and the conserved sequence of 56kDa outer membrane protein gene of Orientia tsutsugamushi had the nucleotide sequence shown in SEQ ID NO. 1.
SEQ ID NO.1：
AGAGCAGAGCTAGGGTTTATGTACCTTAGAAATATAAGCGCTGAGGTTGAAGTAGGTAAAGGCAAGGTTGAAGTAGGTAAAGGTAAGGCAGATTCTGGAGGTGAGATAAAGGCAGATTCTGGAGGTGGGACAGATGCTCCTATTATACGTAAGCGGTTTAAACTTACACCTCCTCAGCCTACTATAATGCCTATAAGTATAGCTGATCGTGACCTTGCGGTTGATATTCCTAACATACCTCAGGCGCAAGCGCAAGCTGCAAATCCTCAGCTTGATCATGAGCAACGTGCTGCAGCTAGGATCGCTTGGTTAAAGAATTGTGCTGGTATTGACTATAGGGTAAAAGATCCTAATAATCTTAATGGGCCTATGGTTATAAATCCGGTGTTGTTAAATATTCCACAGGGTAACCCTAATCCTGTTGGAAATCCACCGCAGCGAGCAAATCAGCCTGCAGATTTTGCGATACATAACCATGAGCAATGGAGGTATATGGTAATTGGACTTGCTGCATTATCAAATGCTAATAAACCTATCGATCCTCCTGTCAAAGTATTAACTGATGAAATTACTCAGAGGAAGGATGAAGTAAGGCCACATGCCGATATAGTCGGTATTGAAGTTCCTAATACTGCTTTGCCTAATAGTGCATCTGTCGAACAGATACAGAATAAAATGCAAGAATTAGGCGAGGTATTAGAAGAGCTCAGAGATTTTTTTGATGGGTATA。
The forward primer of the primers has an oligonucleotide sequence shown in SEQ ID NO.2, the reverse primer has an oligonucleotide sequence shown in SEQ ID NO.3, and the 5' end is marked with Biotin (Biotin).
SEQ ID NO.2：5’ GGATCGCTTGGTTAAAGAATTGTGCTGGTA 3’
SEQ ID NO.3：5’ CCTTCCTCTGAGTAATTTCATCAGTTAATA 3’ 。
An RPA probe for detecting orientia tsutsugamushi was designed based on the sequence of 56kDa outer membrane protein gene of orientia tsutsutsugamushi (GenBank: KM 115577), the conserved sequence of 56kDa outer membrane protein gene of orientia tsugamushi has the nucleotide sequence shown in SEQ ID NO. 1. The probe is positioned at nucleotide 397 to 442 of a 56kDa outer membrane protein gene, has an oligonucleotide sequence shown in SEQ ID No.4, is labeled with fluorescein at the 5 'end, is added with an extension blocking group at the 3' end, and is added with Tetrahydrofuran (THF) between 30 th and 31 th bases.
SEQ ID NO.4：
5’ ATTCCACAGGGTAACCCTAATCCTGTTGGAATCCACCGCAGCGAG 3’ 。
The probe consists of fluorescein, a 5 ' terminal sequence, Tetrahydrofuran (THF), a 3 ' terminal sequence and a 3 ' terminal extension blocking group.
The fluorescein is carboxyl fluorescein FAM or other fluorescein such as FITC, and the extension blocking group is a phosphate group or other blocking groups.
A method of RPA for detecting orientia tsutsugamushi, comprising using primers SEQ ID NO.2, SEQ ID NO.3 and probe SEQ ID NO. 4.
The reagent for 50. mu.L of RPA reaction system in the RPA method for detecting orientia tsutsugamushi, the concentration of the forward primer is 10. mu. mol/L, the concentration of the reverse primer is 10. mu. mol/L, the concentration of the probe is 5. mu. mol/L, the concentration of magnesium ion is 280. mu. mol/L, and the sample addition amount of the template is 1. mu.L. A premix of 2.1. mu.L of the forward primer, 2.1. mu.L of the reverse primer, 0.6. mu.L of the probe, 1. mu.L of the sample, 12.2. mu.L of DNase-free and RNase-free water and 29.5. mu.L of buffer was added to a 0.2mL TwistAmp nfo reaction tube containing lyophilized enzyme powder, and then 2.5. mu.L of a magnesium acetate solution was added to the cap of the reaction tube. Throwing the magnesium acetate solution on the cover of the reaction tube, fully and uniformly mixing, amplifying at 37 ℃ for 20min, taking out the reaction tube in the 4 th min of reaction, fully and uniformly mixing, and then putting the reaction tube back to the reaction device for continuous amplification. The sample adding amount is 5 mu L during detection, and the color development time of the test strip is controlled within 3-5 min.
It is a second object of the present invention to provide a probe for detecting orientia tsutsugamushi. The probe is designed according to the conserved gene 56kDa outer membrane protein of the Orientia tsutsutsugamushi, and the comparison and analysis of the homologous sequence of the gene are carried out by software, so as to further determine the conserved region of the 56kDa outer membrane protein gene of the Orientia tsutsugamushi, wherein the region contains a nucleotide fragment of 730 bases and has the nucleotide sequence shown in SEQ ID NO. 1.
Preferably, the probe consists of several parts of fluorescein (e.g., carboxyfluorescein FAM), 5 ' terminal sequence, Tetrahydrofuran (THF), 3 ' terminal sequence, and 3 ' terminal extension blocking group (e.g., phosphate group).
Preferably, the length of the probe is 45bp, wherein the 5 'end is 30bp, and the 3' end is 15 bp.
Preferably, the probe has an oligonucleotide sequence shown in SEQ ID NO.4, the 5 'end is labeled with fluorescein FAM or FITC, the 3' end is added with an extension blocking group (such as a phosphate group), and Tetrahydrofuran (THF) is added between the 30 th base and the 31 th base. The probe is annealed with amplified DNA marked with Biotin, and the nfo endonuclease in the RPA system cuts off the probe at a THF position, so that the probe can continue to extend at the 3' end under the action of polymerase, and finally, an amplification product double-marked by FAM and Biotin is obtained.
The third purpose of the invention is to provide an RPA detection method for rapid detection of Orientia tsutsugamushi, which adopts the RPA primer and probe to carry out amplification and combines a lateral chromatography nucleic acid detection test strip (hybrid 2T, Milenia Biotec GmbH, Germany) to carry out visual judgment.
The RPA method for detecting orientia tsutsugamushi of the present invention comprises the steps of:
(1) taking the genome DNA of a sample to be detected as a template, and carrying out RPA reaction under the marks of the primer group and the probe;
(2) and (5) judging a result: detecting the RPA product by using the lateral chromatography nucleic acid detection test strip, wherein a detection line and a quality control line are both displayed, and a judgment result is a positive result; the detection line is not shown, the quality control line is shown, and the judgment result is a negative result; the quality control line is not shown, and whether the detection line is shown or not is judged that the result is invalid.
Preferably, the RPA method for detecting orientia tsutsugamushi according to the present invention comprises the following specific steps:
(1) amplification reagent preparation and loading: mu.l of forward primer 2.1. mu.l, 10. mu. mol/L of reverse primer 2.1. mu.l, 5. mu. mol/L of probe 0.6. mu.l, 1. mu.l of sample to be tested, 12.2. mu.l of DNase-free and RNase-free water and 29.5. mu.l of buffer solution were combined to a 0.2mL TwistAmp nfo reaction tube containing lyophilized enzyme powder. Then 2.5. mu.L of magnesium acetate solution was added to the cap of the reaction tube.
(2) Amplification: throwing the magnesium acetate solution on the cover of the reaction tube, fully and uniformly mixing, amplifying at 37 ℃ for 20min, taking out the reaction tube in the 4 th min of reaction, fully and uniformly mixing, and then putting the reaction tube back to the reaction device for continuous amplification.
(3) And (5) judging a result: diluting 5 μ L of RPA amplification product to 100 μ L with PBST, detecting the RPA product with the lateral chromatography nucleic acid detection test strip, wherein the detection line and the quality control line are both shown, and the judgment result is positive; the detection line is not shown, the quality control line is shown, and the judgment result is a negative result; the quality control line is not shown, and whether the detection line is shown or not is judged that the result is invalid.
The principle of the present invention is to detect the specific conserved target sequence of orientia tsutsugamushi, i.e., the conserved sequence of 56kDa outer membrane protein of orientia tsutsugamushi, which can be one of the marker genes of orientia tsutsugamushi, by using the RPA technique.
The RPA method for detecting Orientia tsutsugamushi of the present invention saves the detection time of Orientia tsutsutsugamushi, the detection can be completed in 20min at 37 deg.C, the whole detection process can be completed in 1 hour, and the detection time is greatly shortened compared with conventional PCR and real-time fluorescent quantitative PCR which require several hours.
The RPA method for detecting the orientia tsutsugamushi reduces the reaction temperature, and the RPA can complete the experiment only by keeping the temperature at 37 ℃, which is far lower than 60-95 ℃ of the fluorescence quantitative PCR and 63 ℃ of LAMP.
The RPA method for detecting orientia tsutsugamushi of the present invention is simpler and more portable: the enzyme and other necessary matters required by amplification can be stored in a freeze-drying way, the enzyme and other necessary matters can be placed for a long time at normal temperature, only hydrolysis buffer solution, primers, probes and templates need to be added during amplification, magnesium ions are added for initiating reaction, and the sample does not need to be subjected to complex reaction.
The RPA method for detecting orientia tsutsugamushi of the present invention has high sensitivity and strong specificity. Can be used for on-site or bedside detection and has wide application prospect.
Drawings
The invention will be further explained with reference to the drawings, in which:
FIG. 1 shows that each group of combinations of detection results of different primer combinations in the screening of the optimal primer combination of the RPA detection system is provided with a group of negative controls, the test results are shown in the figure, the upper group is 1-12 groups of positive control samples, and the lower group is the negative control of each group;
FIG. 2 is a graph showing the determination of the optimum reverse primer and probe combination concentration for the detection method of Oriental RPA of tsutsugamushi disease. Setting the concentration gradient of reverse primers as 10 mu mol/L, 5 mu mol/L and 2.5 mu mol/L, setting the concentration of probes as 10 mu mol/L, 5 mu mol/L and 2.5 mu mol/L, combining the reverse primers with three concentrations with the probes with three concentrations respectively to form 9 groups, wherein the combination numbers are shown in Table 3, each group of combination is provided with a group of negative controls, the test results are shown in the figure, and NC represents the negative controls; (A) 1-3 groups and negative controls for each group; (B) negative controls for groups 4-6 and groups thereof; (C) 7-9 groups and negative controls for each group;
FIG. 3 is a graph showing the determination of the optimum amount of sample addition for the detection method of Oriental RPA of tsutsugamushi disease. Setting the sample adding amount to be 1 muL, 2 muL and 5 muL, and respectively setting a negative control for each group of samples, wherein the test result is shown in the figure, and NC represents the negative control;
FIG. 4 is a graph showing the determination of the optimal amplification time for the detection method of Oriental RPA in tsutsugamushi disease. Setting the amplification time to be 10min, 15min and 20min, and respectively setting a negative control for each group of samples with the amplification time, wherein the test result is shown in the figure, and NC represents the negative control;
FIG. 5 shows the sensitivity of the method for detecting RPA of Orientia tsutsugamushi, in which the synthesized positive plasmid was quantified and diluted to a concentration of 10 in a ten-fold dilution manner4 - 100Taking plasmid DNA of copies/mu L as a template to carry out the test, wherein the test conditions are the optimal test conditions, the test result is shown in the figure, and NC represents negative control;
FIG. 6 shows the specificity of the method for detecting RPA of Oriental tsutsugamushi disease, which uses the genomic DNA of Rickettsia rickettsii, Coxiella burnetii, Rickettsia nigra, Rickettsia sibirica, Staphylococcus aureus, Streptococcus suis as the template to carry out the experiment, and the results are shown in the figure.
Detailed Description
The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. The experimental procedures, for which specific conditions are not indicated in the following examples, are generally carried out according to conventional conditions, for example as described in the molecular cloning instructions, published by Sambrook et al, or according to the manufacturer's recommendations.
The various biological materials described in the examples are obtained by way of experimental acquisition for the purposes of this disclosure and should not be construed as limiting the source of the biological material of the invention. In fact, the sources of the biological materials used are wide and any biological material that can be obtained without violating the law and ethics can be used instead as suggested in the examples.
The RPA primers and probes were synthesized by Nanjing Kingsri Biotech, Inc., and all sequence determination was performed by Nanjing Kingsri Biotech, Inc.
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
Example 1 design and screening of primers and probes for Orientia tsutsugamushi
(1) Design of primers and probes
The inventors analytically determined that the specific sequence in the 56kDa outer membrane protein gene of Orientia tsutsugamushi used in the present invention was the target gene through literature search. A known template gene sequence, namely a nucleotide sequence shown in SEQ ID NO.1, is obtained from an NCBI database, and the sequence is synthesized by Nanjing Kingsler Biotech limited to be used as a positive plasmid and used as a template in the processes of subsequent primer probe screening, reaction system optimization and the like. According to the principle of RPA primer and probe design, 7 primers and 1 probe were designed as shown in Table 1.
TABLE 1 primers and probes
 |
(2) Primer screening
A positive plasmid containing the sequence shown in SEQ ID NO.1 of the 56kDa outer membrane protein gene of Orientia tsutsutusgamushi was artificially synthesized, and primers and probes were comprehensively combined into 12 sets of primer combinations with the combination numbers shown in Table 2, using this plasmid as a template. The 12 primer combinations are respectively used for RPA amplification at 37 ℃, the condition shown by the detection line of the lateral chromatography nucleic acid detection test strip is taken as an index, and the primer probe combination with the highest amplification efficiency at 37 ℃ is screened out for evaluation and application of subsequent RPA detection.
A50. mu.L RPA reaction was selected as follows: 2.1. mu.L of forward primer 10. mu. mol/L, 2.1. mu.L of reverse primer 10. mu. mol/L, 0.6. mu.L of probe 10. mu. mol/L, 1X 10101 mu L of copies/mu L of template, 12.2 mu L of DNase-free and RNase-free water and 29.5 mu L of buffer solution to form a premixed solution, and the premixed solution is added into 0.2mL of TwistAmp nfo reaction tubes containing freeze-dried enzyme powder. Then 2.5 mul of magnesium acetate solution is added on the cover of the reaction tube, considering the condition that the RPA reaction sensitivity is higher and false positive is easy to appear, the inventor sets a group of negative control for each group of primer probe combination, the negative control does not add template, and the volume of the template is complemented by water. The inventor also sets a group of positive controls, the positive controls are provided by TwistAmp RPA nfo reagent boxes, meanwhile, a group of negative controls are set for the positive controls, the negative controls are not added with templates, and the volume of the templates is complemented with water. Amplification: throwing the magnesium acetate solution on the cover of the reaction tube, fully and uniformly mixing, amplifying at 37 ℃ for 20min, taking out the reaction tube in the 4 th min of reaction, fully and uniformly mixing, and then putting the reaction tube back to the reaction device for continuous amplification. And (5) judging a result: mu.L of RPA amplification product was diluted to 100. mu.L with PBST and the RPA product was detected using the lateral flow nucleic acid detection strip described above. One multiple well was set for each reaction.
TABLE 2 primer Probe combination numbering
The results of the colloidal gold assay for the 12 primer sets are shown in FIG. 1. FIG. 1 is a diagram showing the detection line and quality control line of a lateral chromatography nucleic acid detection test strip with 12 groups of probe and probe combinations, positive controls and corresponding negative controls when the detection time is 5 min.
The primer combination determined by the invention comprises: two forward primers OtF299 and OtR583, respectively having the oligonucleotide sequences shown in SEQ ID NO.2 and SEQ ID NO. 3.
(3) Determination of the Probe
The OtProbe probes listed in Table 1 are preferred in the present invention, and consist of fluorescein (e.g., carboxyfluorescein FAM), 5 ' terminal sequence, Tetrahydrofuran (THF), 3 ' terminal sequence, and 3 ' terminal extension blocking group (e.g., phosphate group). The length of the probe is 45bp, wherein the 5 'end is 30bp, and the 3' end is 15 bp.
The probe has an oligonucleotide sequence shown in SEQ ID NO.4, the 5 'end is marked with fluorescein FAM, the 3' end is added with an extension blocking group (such as a phosphate group), and Tetrahydrofuran (THF) is added between the 30 th base and the 31 th base. The probe is annealed with amplified DNA marked with Biotin, and the nfo endonuclease in the RPA system cuts off the probe at a THF position, so that the probe can continue to extend at the 3' end under the action of polymerase, and finally, an amplification product double-marked by FAM and Biotin is obtained.
Example 2: optimization of RPA reaction systems, amplification and detection conditions
In the process of primer screening, the lateral chromatography nucleic acid detection test strip still has false positive in detection, so that the RPA reaction system, the amplification and detection conditions need to be optimized
(1) Concentration of primer Probe
Setting the concentration gradient of the reverse primer as 10 mu mol/L, 5 mu mol/L and 2.5 mu mol/L, setting the concentration of the probe as 10 mu mol/L, 5 mu mol/L and 2.5 mu mol/L, combining the reverse primers with three concentrations with the probes with two concentrations respectively to form 6 groups of combinations, and setting a group of negative controls in each group of combinations. Respectively carrying out RPA amplification at 37 ℃, and taking the condition shown by the detection line of the lateral chromatography nucleic acid detection test strip as an index after amplification. The combination with the best amplification effect and no false positive is screened out.
TABLE 3 combination numbering of reverse primer concentrations and Probe concentrations
The analysis of the test result of the lateral chromatography nucleic acid test strip is shown in FIG. 2, in order to make the picture clearly show, A, B, C pieces are divided, and the concentration of the reverse primer determined by the invention is 10 mu mol/L and the concentration of the probe is 5 mu mol/L in consideration of economic factors.
(2) Sample addition amount
Adjusting a reaction system: 2.1. mu.L of forward primer 10. mu. mol/L, 2.1. mu.L of reverse primer 10. mu. mol/L, 0.6. mu.L of probe 5. mu. mol/L, 1X 1041 mu L of copies/mu L of template, 12.2 mu L of DNase-free and RNase-free water and 29.5 mu L of buffer solution to form a premixed solution, and the premixed solution is added into 0.2mL of TwistAmp nfo reaction tubes containing freeze-dried enzyme powder. Then 2.5. mu.L of magnesium acetate solution was added to the lid of the reaction tube, negative control without template, and template volume was made up with water. Amplification: throwing the magnesium acetate solution on the cover of the reaction tube, fully and uniformly mixing, amplifying at 37 ℃ for 20min, taking out the reaction tube in the 4 th min of reversal, fully and uniformly mixing, and then putting the reaction tube back to the reaction device for continuous amplification. And after amplification, the RPA product is detected by using the lateral chromatography nucleic acid detection test strip. The sample adding amount is set to be 1 mu L, 2 mu L and 5 mu L during detection, and each group is provided with a group of negative controls.
As shown in FIG. 3, no false positive occurs in the sample addition amounts of 1. mu.L, 2. mu.L and 5. mu.L, but the sample detection lines of 1. mu.L and 2. mu.L are shallow, and the sample addition amount determined by the present invention is 5. mu.L in consideration of the subsequent sensitivity test.
(3) Time of amplification
A50 μ L RPA reaction was as follows: 2.1. mu.L of forward primer 10. mu. mol/L, 2.1. mu.L of reverse primer 10. mu. mol/L, 0.6. mu.L of probe 5. mu. mol/L, 1X 1041 mu L of copies/mu L of template, 12.2 mu L of DNase-free and RNase-free water and 29.5 mu L of buffer solution to form a premixed solution, and the premixed solution is added into 0.2mL of TwistAmp nfo reaction tubes containing freeze-dried enzyme powder. Then 2.5. mu.L of magnesium acetate solution was added to the cap of the reaction tube. Amplification: throwing the magnesium acetate solution on the cover of the reaction tube, fully and uniformly mixing, amplifying at 37 ℃ for 10min, 15min and 20min, reacting for 4min, taking out the reaction tube, fully and uniformly mixing, and then putting back to the reaction device for continuous amplification. The inventor sets a group of negative controls for each group, the negative controls do not add a template, and the volume of the template is complemented by water. And (5) judging a result: mu.L of RPA amplification product was diluted to 100. mu.L with PBST and the RPA product was detected using the lateral flow nucleic acid detection strip described above.
By analyzing the detection result of the lateral chromatography nucleic acid detection test strip, the amplification time of 10min and 15min is shorter, and the amplification time determined by the invention is 20min in consideration of subsequent sensitivity tests.
In conclusion, through optimization of the RPA reaction system, amplification and detection conditions, the finding shows that the detection effect is best when a forward primer with a concentration of 10 μ M, a reverse primer with a concentration of 10 μ M and a probe with a concentration of 5 μ M are used for amplification for 20min, the sample loading amount is 5 μ L, and the color development time of a test strip is controlled to be 3-5 min.
Example 3: sensitivity evaluation of RPA detection
The Ot-positive plasmid was diluted 10-fold to 104To a series of different concentrations such as 1/. mu.L, 1. mu.L of each of the primers was added to the reaction system determined in example 2, and the selected primer combination was used to perform RPA detection on the template having different copy numbers using the amplification and detection conditions determined in example 2, thereby observing the sensitivity of the RPA detection.
The results are shown in FIG. 5, which shows that the above samples are positive from 10 copies/. mu.L, indicating that the sensitivity of the RPA detection method of the present invention reaches 10 copies/. mu.L.
Example 4: evaluation of specificity of RPA detection
The specific evaluation takes the genomic DNA of rickettsia immediately, coxiella burnetii, rickettsia blacklongriver, rickettsia sibirica, staphylococcus aureus and streptococcus suis as a reference to determine the specificity of the RPA detection method.
Using genome DNAs of Oriental tsutsugamushi, rickettsia rickettsii, coxiella burnetii, rickettsia fulgidae, rickettsia sibirica, staphylococcus aureus and streptococcus suis as templates, respectively, and adopting the following reaction systems: mu.l of forward primer 2.1. mu.l, 10. mu. mol/L of reverse primer 2.1. mu.l, 5. mu. mol/L of probe 0.6. mu.l, 1. mu.l of sample, 12.2. mu.l of DNase-free and RNase-free water and 29.5. mu.l of buffer make up a premix, which was added to a 0.2mL TwistAmp nfo reaction tube containing lyophilized enzyme powder. Then 2.5. mu.L of magnesium acetate solution was added to the cap of the reaction tube. Amplification: throwing the magnesium acetate solution on the cover of the reaction tube, fully and uniformly mixing, amplifying at 37 ℃ for 20min, and taking out the reaction tube for fully and uniformly mixing at the reaction 4 min. And (5) judging a result: diluting 5 μ L of RPA amplification product to 100 μ L with PBST, detecting the RPA product with the lateral chromatography nucleic acid detection test strip, wherein the detection line and the quality control line are both shown, and the judgment result is positive; the detection line is not shown, the quality control line is shown, and the judgment result is a negative result; the quality control line is not shown, and whether the detection line is shown or not is judged that the result is invalid.
The results are shown in figure 6, the genome DNA sample detection lines of rickettsia immediately afterwards, coxiella burnetii, siberian rickettsia, staphylococcus aureus and streptococcus suis do not have a stripe, and the detection lines are negative, and only the detection lines of oriental tsugamushi disease samples have a clear stripe and are positive, which shows that the RPA detection method of the invention has strong specificity to oriental tsugamushi disease.
Sequence listing
<110> Liviehig
<120> a method for detecting RPA of orientia tsutsugamushi, a primer and a probe therefor, and use thereof
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 730
<212> DNA
<213> Orientia tsutsutsugamushi (Orientia tsutsutsugamushi)
<220>
<221> misc_feature
<222> (1)..(730)
<400> 1
agagcagagc tagggtttat gtaccttaga aatataagcg ctgaggttga agtaggtaaa 60
ggcaaggttg aagtaggtaa aggtaaggca gattctggag gtgagataaa ggcagattct 120
ggaggtggga cagatgctcc tattatacgt aagcggttta aacttacacc tcctcagcct 180
actataatgc ctataagtat agctgatcgt gaccttgcgg ttgatattcc taacatacct 240
caggcgcaag cgcaagctgc aaatcctcag cttgatcatg agcaacgtgc tgcagctagg 300
atcgcttggt taaagaattg tgctggtatt gactataggg taaaagatcc taataatctt 360
aatgggccta tggttataaa tccggtgttg ttaaatattc cacagggtaa ccctaatcct 420
gttggaaatc caccgcagcg agcaaatcag cctgcagatt ttgcgataca taaccatgag 480
caatggaggt atatggtaat tggacttgct gcattatcaa atgctaataa acctatcgat 540
cctcctgtca aagtattaac tgatgaaatt actcagagga aggatgaagt aaggccacat 600
gccgatatag tcggtattga agttcctaat actgctttgc ctaatagtgc atctgtcgaa 660
cagatacaga ataaaatgca agaattaggc gaggtattag aagagctcag agattttttt 720
gatgggtata 730
<210> 2
<211> 30
<212> DNA
<213> Orientia tsutsutsugamushi (Orientia tsutsutsugamushi)
<220>
<221> misc_feature
<222> (1)..(30)
<400> 2
ggatcgcttg gttaaagaat tgtgctggta 30
<210> 3
<211> 30
<212> DNA
<213> Orientia tsutsutsugamushi (Orientia tsutsutsugamushi)
<220>
<221> misc_feature
<222> (1)..(30)
<223> 5' end labeling biotin
<400> 3
ccttcctctg agtaatttca tcagttaata 30
<210> 4
<211> 45
<212> DNA
<213> Probe Otprobe (Orientia tsutsutsugamushi) within 56kDa outer Membrane protein Gene of Orientia
<220>
<221> misc_feature
<222> (1)..(45)
<223> fluorescein FAM labeled at 5 'end, extension blocking group (e.g., phosphate group) added at 3' end, and Tetrahydrofuran (THF) inserted between bases at positions 30-31
<400> 4
attccacagg gtaaccctaa tcctgttgga atccaccgca gcgag 45
Claims (4)
1. An RPA special primer and probe for detecting Orientia tsutsutsugamushi is designed based on the conserved sequence of 56kDa outer membrane protein gene of Orientia tsutsutsugamushi, said conserved sequence of 56kDa outer membrane protein gene of Orientia tsutsugamushi is the nucleotide sequence shown in SEQ ID NO. 1;
SEQ ID NO.1：
AGAGCAGAGCTAGGGTTTATGTACCTTAGAAATATAAGCGCTGAGGTTGAAGTAGGTAAAGGCAAGGTTGAAGTAGGTAAAGGTAAGGCAGATTCTGGAGGTGAGATAAAGGCAGATTCTGGAGGTGGGACAGATGCTCCTATTATACGTAAGCGGTTTAAACTTACACCTCCTCAGCCTACTATAATGCCTATAAGTATAGCTGATCGTGACCTTGCGGTTGATATTCCTAACATACCTCAGGCGCAAGCGCAAGCTGCAAATCCTCAGCTTGATCATGAGCAACGTGCTGCAGCTAGGATCGCTTGGTTAAAGAATTGTGCTGGTATTGACTATAGGGTAAAAGATCCTAATAATCTTAATGGGCCTATGGTTATAAATCCGGTGTTGTTAAATATTCCACAGGGTAACCCTAATCCTGTTGGAAATCCACCGCAGCGAGCAAATCAGCCTGCAGATTTTGCGATACATAACCATGAGCAATGGAGGTATATGGTAATTGGACTTGCTGCATTATCAAATGCTAATAAACCTATCGATCCTCCTGTCAAAGTATTAACTGATGAAATTACTCAGAGGAAGGATGAAGTAAGGCCACATGCCGATATAGTCGGTATTGAAGTTCCTAATACTGCTTTGCCTAATAGTGCATCTGTCGAACAGATACAGAATAAAATGCAAGAATTAGGCGAGGTATTAGAAGAGCTCAGAGATTTTTTTGATGGGTATA ；
the forward primer in the primer is shown as the oligonucleotide sequence shown in SEQ ID NO.2, the reverse primer is shown as the oligonucleotide sequence shown in SEQ ID NO.3, and the 5' end is marked with Biotin (Biotin);
SEQ ID NO.2：5’ GGATCGCTTGGTTAAAGAATTGTGCTGGTA 3’，
SEQ ID NO.3：5’ CCTTCCTCTGAGTAATTTCATCAGTTAATA 3’ ；
the probe has an oligonucleotide sequence shown as SEQ ID NO.4, fluorescein is marked at the 5 'end, an extension blocking group is added at the 3' end, and Tetrahydrofuran (THF) is added between the 30 th and 31 th bases;
SEQ ID NO.4：
5’ ATTCCACAGGGTAACCCTAATCCTGTTGGAATCCACCGCAGCGAG 3’。
2. the primers and probes according to claim 1, characterized in that: the fluorescein is FAM or FITC, and the extension blocking group is a phosphate group.
3. Use of the primer and probe as set forth in any one of claims 1-2 for the preparation of RPA detection reagent of orientia tsutsugamushi.
4. Use according to claim 3, characterized in that: the reagent is used for a 50 mu L RPA reaction system in the application, the concentration of the forward primer is 10 mu mol/L, the concentration of the reverse primer is 10 mu mol/L, the concentration of the probe is 5 mu mol/L, the concentration of magnesium ions is 280 mu mol/L, and the sample adding amount of the template is 1 mu L; a premix of 2.1. mu.L of the forward primer, 2.1. mu.L of the reverse primer, 0.6. mu.L of the probe, 1. mu.L of the sample, 12.2. mu.L of DNase-free and RNase-free water and 29.5. mu.L of buffer was added to a 0.2mL TwistAmp nfo reaction tube containing lyophilized enzyme powder, and then 2.5. mu.L of a magnesium acetate solution was added to the cap of the reaction tube; throwing the magnesium acetate solution on the cover of the reaction tube, fully and uniformly mixing, amplifying at 37 ℃ for 20min, taking out the reaction tube for fully and uniformly mixing in the 4 th min of reaction, and then putting the reaction tube back to the reaction device for continuous amplification; the sample adding amount is 5 mu L during detection, and the color development time of the test strip is controlled within 3-5 min.
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| CN115232798B (en) * | 2022-08-08 | 2023-05-02 | 南京工业大学 | Hybridoma cell strain 5B3, orientia tsutsugamushi 56kDa protein monoclonal antibody, and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100514438B1 (en) * | 2003-05-22 | 2005-09-14 | 대한민국 | A recombinant protein useful for the detection of tsutsugamushi infection, a gene encoding the same, and a diagnostic kit using the same |
| CN103866013A (en) * | 2014-03-06 | 2014-06-18 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Orientia tsutsugamushi disease nucleic acid detection kit |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100514438B1 (en) * | 2003-05-22 | 2005-09-14 | 대한민국 | A recombinant protein useful for the detection of tsutsugamushi infection, a gene encoding the same, and a diagnostic kit using the same |
| CN103866013A (en) * | 2014-03-06 | 2014-06-18 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Orientia tsutsugamushi disease nucleic acid detection kit |
Non-Patent Citations (2)
| Title |
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| Development of Recombinase Polymerase Amplification Assays for Detection of Orientia tsutsugamushi or Rickettsia typhi;Chao Chien-Chung等;《PLoS neglected tropical diseases》;20150710;第9卷(第7期);摘要，第4页第2段，第6页第3段，第7页3段，第8页第2段，图1-2，表1 * |
| 环介质等温扩增技术快速检测恙虫病东方体的研究;任立松等;《现代预防医学》;20111025;第38卷(第20期);摘要，第1.3节 * |
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