Primer probe combination, kit and method for detecting rickettsia pathogen

Abstract

The invention relates to the technical field of gene detection, and discloses a primer probe combination, a kit and a method for detecting rickettsia pathogens. The rickettsia pathogens comprise orientia tsutsugamushi and rickettsia przewalskii; the invention specifically discloses a primer probe combination of orientia tsutsugamushi and a primer probe combination of rickettsia przewalskii. According to the invention, a nestMIRA technology is utilized, and specific primers and probes are designed aiming at a groEL gene of orientia tsutsugamushi and an ompB gene of rickettsia przewalskii. The primer probe combination, the kit and the method for detecting the rickettsia pathogen have the characteristics of strong specificity and high sensitivity, and the lower detection limits of the primer probe combination, the kit and the method respectively reach 1.64 copies / [mu] L and 1.28 copies / [mu] L. The method is simple and convenient to operate, does not need complex equipment, can quickly complete amplification reaction under a constant-temperature condition, and is suitable for clinical quick diagnosis and field detection.

Description

Technical Field

[0001] The present invention relates to the field of gene detection technology, and in particular to a primer-probe combination, a kit and a method for detecting rickettsial pathogens. Background Art

[0002] Orientia tsutsugamushi and Rickettsia prowazekii are two important rickettsial pathogens that cause scrub typhus (tsutsugamushi) and epidemic typhus. These pathogens are intracellular parasites with complex pathogenic mechanisms and enter humans primarily through the bites of chigger larvae and transmission by body lice.

[0003] In Asia, including China, Japan, South Korea and Southeast Asia, scrub typhus and epidemic typhus are widespread, especially during the rainy season. People infected with scrub typhus usually show high fever, rash and swollen lymph nodes, which may lead to multiple organ failure in severe cases. Patients with epidemic typhus have high fever, headache, rash and central nervous system damage as the main clinical features. The two diseases show similar symptoms in the acute phase, which makes clinical diagnosis difficult and poses a challenge to timely treatment of patients.

[0004] At present, clinical detection of Orientia tsutsugamushi and Rickettsia prowazekii mainly relies on serological tests and molecular biological methods. Although serological detection methods such as indirect immunofluorescence, enzyme-linked immunosorbent assay, and Weil-Felix test can detect antibody levels in serum, these methods are complicated and time-consuming, and may miss diagnoses in chronically infected people with low antibody levels. Molecular biological methods, such as PCR (polymerase chain reaction), can achieve early diagnosis by detecting the DNA of pathogens, but they require large equipment and complex operations, which limits their application in rapid on-site screening. Therefore, it is urgent to develop rapid, simple and accurate detection methods to play a role in the early diagnosis and control of such infections.

[0005] With the development of molecular biotechnology, technologies such as recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP) have been widely used in pathogen diagnosis. However, the existing detection technologies for Orientia tsutsugamushi and Rickettsia prowazekii have technical problems such as long detection time, poor specificity, and low sensitivity, which have not been effectively solved. Multi-enzyme isothermal rapid amplification (MIRA) has the advantages of high detection sensitivity, strong specificity, low cost, and fast speed, providing a new direction for the development of rapid, simple, and accurate detection methods for these two pathogens. This progress not only helps early diagnosis and treatment, but also plays a key role in public health prevention and control. Summary of the invention

[0006] In view of the problems and current status of the prior art, the present invention applies the nestMIRA technology to the detection field of Orientia tsutsugamushi and Rickettsia prowazekii. The purpose of the present invention is to use the nestMIRA technology to achieve rapid, specific and sensitive detection of Orientia tsutsugamushi and Rickettsia prowazekii nucleic acids.

[0007] To achieve the above object, the present invention provides the following technical solutions: In a first aspect, the present invention provides a primer-probe combination for detecting rickettsial pathogens, including Orientia tsutsugamushi and Rickettsia prowazekii; The primer-probe combination of the groEL gene of Orientia tsutsugamushi, the inner upstream primer sequence is shown in SEQ ID NO:4, the inner downstream primer sequence is shown in SEQ ID NO:6, the outer upstream primer sequence is shown in SEQ ID NO:10, the outer downstream primer sequence is shown in SEQ ID NO:12, the fluorescent detection probe sequence is shown in SEQ ID NO:15, and the colloidal gold detection probe sequence is shown in SEQ ID NO:16; The primer-probe combination for the ompB gene of Rickettsia prowazekii, the inner upstream primer sequence is shown in SEQ ID NO: 17, the inner downstream primer sequence is shown in SEQ ID NO: 22, the outer upstream primer sequence is shown in SEQ ID NO: 24, the outer downstream primer sequence is shown in SEQ ID NO: 28, the fluorescent detection probe sequence is shown in SEQ ID NO: 30, and the colloidal gold detection probe sequence is shown in SEQ ID NO: 31.

[0008] The primer-probe combination used for the fluorescence detection of Orientia tsutsugamushi includes 4 primers (SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:12) and a fluorescence detection probe (SEQ ID NO:15); the primer-probe combination used for the colloidal gold test strip detection includes 4 primers (SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14) and a colloidal gold detection probe (SEQ ID NO:16).

[0009] The primer-probe combination used for fluorescence detection of Rickettsia prowazekii includes 4 primers (SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 28) and a fluorescence detection probe (SEQ ID NO: 30); the primer-probe combination used for colloidal gold test strip detection includes 4 primers (SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 29) and a colloidal gold detection probe (SEQ ID NO: 31); Furthermore, the Orientia tsutsugamushi fluorescent detection probe (sequence: SEQ ID NO: 15) is labeled with FAM at the 27th base from the 5' end, the 28th base is replaced by tetrahydrofuran (THF), the 30th base is labeled with BHQ1, and the 3' end is modified by C3spacer blocking; the Rickettsia prowazekii fluorescent detection probe (sequence: SEQ ID NO: 30) is labeled with FAM at the 34th base from the 5' end, the 35th base is replaced by tetrahydrofuran (THF), the 37th base is labeled with BHQ1, and the 3' end is modified by C3spacer blocking; The colloidal gold test strip detection probe for Orientia tsutsugamushi (SEQ ID NO: 16) has a FAM group modified at the 5' end, the 28th base from the 5' end is replaced by tetrahydrofuran (THF), and the 3' end is modified by C3spacer blocking; the colloidal gold test strip detection probe for Rickettsia prowazekii (SEQ ID NO: 31) has a Dig group modified at the 5' end, the 35th base from the 5' end is replaced by tetrahydrofuran (THF), and the 3' end is modified by C3spacer blocking; The 5' ends of the Orientia tsutsugamushi downstream primer sequence (SEQ ID NO:6) and the Rickettsia prowazekii downstream primer (SEQ ID NO:21) are labeled with Biotin, and the sequences after labeling are shown in SEQ ID NO:14 and SEQ ID NO:29.

[0010] In a second aspect, the present invention provides the use of a primer-probe combination in preparing a kit for detecting Orientia tsutsugamushi and Rickettsia prowazekii.

[0011] In a third aspect, the present invention provides a kit for detecting Orientia tsutsugamushi and Rickettsia prowazekii, wherein the kit contains the above-mentioned primer-probe combination.

[0012] In a fourth aspect, the present invention provides a method for detecting Orientia tsutsugamushi and Rickettsia prowazekii based on the nestMIRA technology, wherein the method is not intended for the diagnosis and treatment of the disease, and the method comprises the following steps: Step 1: Extract DNA from the sample to be tested; Step 2: Using the extracted sample DNA as a template, prepare a reaction system containing the above primer-probe combination.

[0013] ; It should be noted that the nestMIRA detection is achieved in two steps. The first-step reaction uses peripheral primers to pre-amplify the target sequence with a reaction time of 10 minutes. After the reaction, the first-step product is added to the second-step reaction system and the target sequence is amplified for the second time using internal primers.

[0014] Step 3: Interpretation of results: ① Fluorescence detection: In the nestMIRA reaction, a fluorescence quantifier or a convenient fluorescence collector can be used to detect the fluorescence signal in real time, and the result can be judged based on the presence or absence of a fluorescence amplification curve; ② Colloidal gold test strip detection: After the nestMIRA reaction is completed, take 10μL of the amplified product and mix it with 70μL ddH2O, add it to the sample well of the colloidal gold test strip, wait for 15 minutes, and interpret the result based on whether there is an obvious red band on the test line (T line).

[0015] The advantages and positive effects of the primer-probe combination, kit and method for detecting Rickettsial pathogens of the present invention are: 1. Based on the nestMIRA technology, the present invention develops a convenient and rapid detection method for detecting Orientia tsutsugamushi and Rickettsia prowazekii with the groEL gene of Orientia tsutsugamushi and the ompB gene of Rickettsia prowazekii as targets. The result detection can be completed in 35 minutes and can be applied to bedside detection.

[0016] 2. The primer probe set of the present invention can achieve efficient and specific nestMIRA amplification and detection. The detection method established thereby has the advantages of high sensitivity, strong specificity and high accuracy. The minimum detection limits for Orientia tsutsugamushi and Rickettsia prowazekii DNA can reach 1.62 copies / μL and 1.28 copies / μL.

[0017] 3. The detection method of the present invention has a short detection time, simple operation steps, no need for large-scale nucleic acid amplification instruments, low detection costs, and greatly saves manpower and material resources. It can achieve rapid and efficient on-site detection of Orientia tsutsugamushi and Rickettsia prowazekii, which is of great significance for the early diagnosis of the disease and the observation of the progression of the disease, thereby guiding clinical medication. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the nestMIRA detection method of Orientia tsutsugamushi and Rickettsia prowazekii in an embodiment of the present invention; Figure 2The results of real-time fluorescence nestMIRA amplification of Orientia tsutsugamushi and Rickettsia prowazekii using different inner primers in the embodiment of the present invention, wherein A is the amplification result of Orientia tsutsugamushi and B is the amplification result of Rickettsia prowazekii; Figure 3 The results of real-time fluorescent nestMIRA amplification of Orientia tsutsugamushi and Rickettsia prowazekii using different peripheral primers in the embodiment of the present invention, wherein A is the amplification result of Orientia tsutsugamushi and B is the amplification result of Rickettsia prowazekii; Figure 4 The results of real-time fluorescence nestMIRA amplification of Orientia tsutsugamushi and Rickettsia prowazekii at different temperatures in the embodiment of the present invention, wherein A is the amplification result of Orientia tsutsugamushi and B is the amplification result of Rickettsia prowazekii; Figure 5 Specificity evaluation of the real-time fluorescence nestMIRA rapid detection method for Orientia tsutsugamushi and Rickettsia prowazekii in the embodiment of the present invention, wherein A is the specific result of Orientia tsutsugamushi and B is the specific result of Rickettsia prowazekii; Figure 6 The figure is a sensitivity evaluation of the real-time fluorescence nestMIRA rapid detection method for Orientia tsutsugamushi and Rickettsia prowazekii in the embodiment of the present invention, wherein A is the sensitivity result of Orientia tsutsugamushi and B is the sensitivity result of Rickettsia prowazekii; Figure 7 This is a sensitivity evaluation of the nestMIRA colloidal gold test strip rapid detection method for Orientia tsutsugamushi and Rickettsia prowazekii in the embodiments of the present invention. DETAILED DESCRIPTION

[0019] The technical solution of the present invention is further described below through the accompanying drawings and embodiments. Unless otherwise defined, the technical terms or scientific terms used in the present invention should have the common meanings understood by people with ordinary skills in the field to which the present invention belongs.

[0020] Unless otherwise defined or described, all professional and scientific terms used in the present invention have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the method of the present invention. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0021] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The experimental methods without specific conditions in the following examples are usually measured in accordance with national standards. The experimental instruments, equipment and reagents without source indication in the following examples are all commercially available raw materials.

[0022] The experimental materials used in the following examples include: Nucleic acids of different pathogens: Orientia tsutsugamushi, Rickettsia prowazekii, Rickettsia moorei, Coxiella burnetii, Anaplasma phagocytophilum, Bartonella, Leptospira, Borrelia burgdorferi, Brucella, Shigella dysenteriae, Salmonella typhimurium, Listeria, Staphylococcus aureus, Streptococcus, Heilongjiang spotted fever Rickettsia, Siberian Rickettsia, Conor Rickettsia, Bartonella, Malaria, Hantavirus, Dengue virus.

[0023] Reagent materials: MIRA amplification kit was purchased from AMP-Future Biotech.

[0024] Plasmids: plasmids containing the groEL gene fragment of Orientia tsutsugamushi and plasmids containing the ompB gene fragment of Rickettsia prowazekii.

[0025] Example 1 Design of nestMIRA primer probe set for targeted detection of Orientia tsutsugamushi and Rickettsia prowazekii In order to obtain the nestMIRA primer-probe combination with the best amplification efficiency, multiple sets of nestMIRA peripheral primer sets, internal primer sets and probes for Orientia tsutsugamushi were designed based on the groEL gene of Orientia tsutsugamushi; multiple sets of nestMIRA peripheral primer sets, internal primer sets and probes for Rickettsia prowazekii were designed based on the ompB gene of Rickettsia prowazekii. The detailed sequence information is shown in Table 1.

[0026] Table 1 Sequence information ; ; The above primers and probes were synthesized by General Biotechnology (Anhui) Co., Ltd.

[0027] Example 2 Establishment of a method for detecting Orientia tsutsugamushi and Rickettsia prowazekii based on nestMIRA technology In this example, three core factors that affect the detection sensitivity of nestMIRA technology were optimized to screen out the best reaction system and reaction conditions. The fluorescence detection was used as an example to show the test results. In this example, the positive plasmid concentration of Orientia tsutsugamushi used was: 1.62×10 4 copies / μL, the concentration of Rickettsia prowazekii positive plasmid is: 1.28×10 4 copies / μL.

[0028] (1) Optimization of nestMIRA inner primers: In Example 1, multiple inner upstream primers and inner downstream primers were designed. They were tested in pairs. There were 12 combinations for Orientia tsutsugamushi and 9 combinations for Rickettsia prowazekii. The MIRA amplification reaction was performed using the amplification kit of AMP-Future Biotech, and the reaction system and procedure are shown in Table 2: Table 2 Reaction system and procedure ; The results of primer screening for Orientia tsutsugamushi and Rickettsia prowazekii were obtained from Figure 2 As shown. Figure 2 The results showed that the optimal primer combination for the inner part of Orientia tsutsugamushi was GroEL-F4+GroEL-R2 (SEQ ID NO.4+SEQ ID NO.6), and the optimal primer combination for the inner part of Rickettsia prowazekii was Ombp-F1+Ombp-R3 (SEQ ID NO.17+SEQ ID NO.22).

[0029] (2) Optimization of nestMIRA peripheral primers: After obtaining the optimal inner primer combination, the designed outer upstream primer and downstream primer were combined. There are 9 combinations for Orientia tsutsugamushi and 9 combinations for Rickettsia prowazekii. The 9 outer primer combinations and the optimal inner primer combination were tested by nestMIRA to screen out the optimal outer primer combination. According to the instructions of the amplification kit of AMP-Future Biotech, the reaction system was prepared as shown in Table 3: Table 3 Reaction system ; Note: nestMIRA consists of a peripheral MIRA reaction + an inner MIRA reaction, which is carried out in two steps. After the peripheral MIRA reaction is completed, 10 μL of the amplified product is added to the inner MIRA reaction system, and the amplification reaction is performed again. The fluorescence signal is collected using a fluorescence quantitative PCR instrument or a convenient fluorescence collector to complete the detection.

[0030] The results of peripheral primer combination screening are as follows Figure 3 As shown, the optimal external primer combination for Orientia tsutsugamushi is: GroEL-WF3 + GroEL-WR2 (SEQ ID NO.10 + SEQ ID NO.12); the optimal external primer combination for Rickettsia prowazekii is: Ombp-WF2 + Ombp-WR3 (SEQ ID NO.24 + SEQ ID NO.28).

[0031] (3) Screening of optimal reaction temperature of nestMIRA: After determining the best peripheral primer combination and inner primer combination, the nestMIRA reaction temperature was screened. Five temperature gradients (37°C, 38°C, 39°C, 40°C, 41°C) were set, the reaction system was configured according to Table 3, and the nestMIRA reaction was performed. The results are shown in Figure 3. Figure 4 As shown, stronger fluorescence signals were obtained at 38°C compared with the other four reaction temperatures, so 38°C was selected as the optimal nestMIRA reaction temperature for Orientia tsutsugamushi and Rickettsia prowazekii.

[0032] Example 3 Evaluation of the specificity of nestMIRA detection method for Orientia tsutsugamushi and Rickettsia prowazekii

[0033] The experiment is carried out according to the best primer combination, the best reaction system and the best reaction conditions screened out.

[0034] Specificity validation of MIRA reaction for Orientia tsutsugamushi and Rickettsia prowazekii was performed using 20 different pathogens, including Orientia tsutsugamushi, Rickettsia prowazekii, Rickettsia morozekii, Coxiella burnetii, Anaplasma phagocytophilum, Bartonella, Leptospira, Borrelia burgdorferi, Brucella, Shigella dysenteriae, Salmonella typhimurium, Listeria, Staphylococcus aureus, Streptococcus, Heilongjiang spotted fever Rickettsia, Siberian Rickettsia, Conor Rickettsia, Bartonella, Malaria, Hantavirus, Dengue virus. Reaction results are as follows Figure 5 As shown by Figure 5 It can be seen that only the target pathogen showed an obvious amplification curve, while the other pathogens and negative controls did not show an amplification curve. The results showed that this detection method had no cross-reaction with the 20 pathogens tested and had strong specificity.

[0035] Example 4 Evaluation of the sensitivity of the nestMIRA detection method for Orientia tsutsugamushi and Rickettsia prowazekii

[0036] The positive plasmid of Orientia tsutsugamushi and the positive plasmid of Rickettsia prowazekii were diluted 10 times in a stepwise manner. The concentration of Orientia tsutsugamushi was: 1.62×10 4 copies / μL, 1.62×10 3 copies / μL, 1.62×10 2 copies / μL, 1.62×10 1 copies / μL, 1.62 copies / μL; Rickettsia prowazekii concentration: 1.28×10 4 copies / μL, 1.28×10 3 copies / μL, 1.28×10 2 copies / μL, 1.28×10 1Six consecutive positive plasmid samples with different concentrations were used as templates to perform nestMIRA reactions to evaluate the sensitivity of the detection method.

[0037] The sensitivity of nestMIRA was evaluated by fluorescence detection and colloidal gold test strips. The results are as follows: Figure 6 and Figure 7 As shown, the detection limit of Orientia tsutsugamushi nestMIRA is 1.62 copies / μL, the detection limit of Rickettsia prowazekii nestMIRA is 1.28 copies / μL, and the fluorescence detection has the same detection limit as the colloidal gold test strip detection, indicating that the method of the present invention has high sensitivity in detecting Orientia tsutsugamushi and Rickettsia prowazekii.

[0038] Example 5 Application of nestMIRA detection method for Orientia tsutsugamushi and Rickettsia prowazekii

[0039] In order to fully evaluate the nestMIRA detection method for Orientia tsutsugamushi and Rickettsia prowazekii, blood samples from 20 suspected typhus patients were collected for nestMIRA testing. The specific steps are as follows: Step 1: Extract DNA from the sample to be tested; Step 2: Using the extracted sample DNA as a template, prepare a reaction system containing the nestMIRA amplification primer probe combination. The reaction system preparation is as described in Example 2.

[0040] Step 3: Interpretation of results: ① Fluorescence detection: In the nestMIRA reaction, a fluorescence quantifier or a convenient fluorescence collector can be used to detect the fluorescence signal in real time, and the result can be judged based on the presence or absence of a fluorescence amplification curve; ② Colloidal gold test strip detection: After the nestMIRA reaction is completed, take 10μL of the amplified product and mix it with 70μL ddH2O, add it to the sample well of the colloidal gold test strip, wait for 15 minutes, and interpret the result based on whether there is an obvious red band on the test line (T line).

[0041] The test results of blood samples from 20 suspected patients are shown in Table 4: Table 4 Blood sample test results ; The test results in Table 4 show that the nestMIRA detection method for Orientia tsutsugamushi and Rickettsia prowazekii established in the present invention is consistent with the qPCR detection results, indicating that the method established in the present invention has good detection performance and is a reliable detection method.

[0042] In summary, the present invention has developed a nestMIRA detection method for detecting Orientia tsutsugamushi and Rickettsia prowazekii based on the nestMIRA technology. This method can be detected by fluorescence detection or by colloidal gold test strips. The detection method has strong specificity and high sensitivity. In particular, the use of colloidal gold test strips can achieve dual pathogen detection, which greatly saves the detection cost. Compared with the fluorescent qPCR technology, this method does not require expensive experimental equipment, is simple to operate, realizes bedside detection of pathogens, and provides a reliable detection technology for early detection of diseases and control of diseases.

[0043] Therefore, the present invention adopts the above-mentioned primer probe combination, kit and method for detecting Rickettsial pathogens, which has the characteristics of strong specificity and high sensitivity, and the detection limits are 1.64 copies / μL and 1.28 copies / μL respectively. The method is easy to operate, does not require complex equipment, can quickly complete the amplification reaction under constant temperature conditions, and is suitable for rapid clinical diagnosis and on-site detection.

[0044] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A primer-probe combination for detecting rickettsial pathogens, characterized in that: Rickettsial pathogens include Orientia tsutsugamushi and Rickettsia prowazekii; The primer-probe combination of Orientia tsutsugamushi, the inner upstream primer sequence is shown in SEQ ID NO:4, the inner downstream primer sequence is shown in SEQ ID NO:6, the outer upstream primer sequence is shown in SEQ ID NO:10, the outer downstream primer sequence is shown in SEQ ID NO:12, the fluorescent detection probe sequence is shown in SEQ ID NO:15, and the colloidal gold detection probe sequence is shown in SEQ ID NO:16; The primer-probe combination for Rickettsia prowazekii, the inner upstream primer sequence is shown in SEQ ID NO: 17, the inner downstream primer sequence is shown in SEQ ID NO: 22, the outer upstream primer sequence is shown in SEQ ID NO: 24, the outer downstream primer sequence is shown in SEQ ID NO: 28, the fluorescent detection probe sequence is shown in SEQ ID NO: 30, and the colloidal gold detection probe sequence is shown in SEQ ID NO:

31.

2. The primer-probe combination according to claim 1, characterized in that: The inner downstream primers shown in SEQ ID NO:6 and SEQ ID NO:21 were modified with Biotin, and the modified sequences were shown in SEQ ID NO:14 and SEQ ID NO:

29.

3. The primer-probe combination according to claim 1, characterized in that: The fluorescent detection probes shown in SEQ ID NO: 15 and SEQ ID NO: 30 are modified with groups including i6FAMdT, idSp, iBHQ1dT and C3-spacer.

4. The primer-probe combination according to claim 3, characterized in that: i6FAMdT is thymidine deoxynucleotide labeled with 6-carboxyfluorescein, idSp is a tetrahydrofuran spacer, iBHQ1dT is a quenching group, and C3-spacer is a blocking group modified with a C3 spacer arm.

5. The primer-probe combination according to claim 1, characterized in that: The colloidal gold detection probe shown in SEQ ID NO: 16 is modified with groups including FAM, idSp, and C3 Spacer; the colloidal gold detection probe shown in SEQ ID NO: 31 is modified with groups including Dig, idSp, and C3 Spacer.

6. Use of the primer-probe combination according to any one of claims 1 to 5 in preparing a kit for detecting Orientia tsutsugamushi and Rickettsia prowazekii.

7. A kit for detecting Orientia tsutsugamushi and Rickettsia prowazekii, characterized in that: A primer-probe combination comprising any one of claims 1 to 5.

8. A method for detecting Orientia tsutsugamushi and Rickettsia prowazekii based on nestMIRA technology, which is not intended for the diagnosis and treatment of the disease, characterized in that: The following steps are involved: Step 1: Extract DNA from the sample to be tested; Step 2: using the extracted sample DNA as a template, preparing a reaction system containing the primer-probe combination according to any one of claims 1 to 5; Step 3: Interpretation of results: ① Fluorescence detection: In the nestMIRA reaction, a fluorescence quantifier or a convenient fluorescence collector is used to detect the fluorescence signal in real time, and the result is determined based on the presence or absence of a fluorescence amplification curve; ② Colloidal gold test strip detection: After the nestMIRA reaction is completed, take 10μL of the amplified product and mix it with 70μL ddH2O, add it to the sample well of the colloidal gold test strip, wait for 15 minutes, and interpret the result based on whether there is an obvious red band on the test line.

9. The method for detecting Orientia tsutsugamushi and Rickettsia prowazekii based on nestMIRA technology according to claim 8, characterized in that: The peripheral reaction temperature and the inner reaction temperature are 37-41°C, the peripheral reaction time is 10 minutes, and the inner reaction time is 20 minutes.

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