Composition for detecting rickettsia pathogen, kit and application

Abstract

The invention belongs to the field of molecular biological detection, particularly relates to rapid detection and identification of rickettsia pathogens, and more particularly relates to detection of five pathogenic pathogens including rickettsia sibirica, rickettsia rickettsia, rickettsia araneus, rickettsia commisonii and Japanese rickettsia through a multiple real-time fluorescent PCR (polymerase chain reaction) detection technology. The composition provided by the invention can realize specific detection of five pathogenic pathogens, has high detection sensitivity (up to 500 copies / mL), can simply, rapidly and accurately detect and distinguish rickettsia pathogens, shortens the judgment time of pathogen types, and accelerates implementation of countermeasures.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biological detection, and specifically, relates to the rapid detection and identification of Rickettsia pathogens, and more specifically, relates to the detection and differentiation of five pathogenic pathogens, namely, Rickettsia sibirica, Rickettsia rickettsii, Rickettsia mitochondrialis, Rickettsia conorii and Rickettsia japonicus, by using multiplex real-time fluorescence PCR detection technology. Background Art

[0002] The genus Rickettsia includes typhus group and spotted fever group Rickettsiae. Typhus group mainly includes Rickettsia prowazekii, which causes epidemic typhus, and Rickettsia morozekii, which causes endemic typhus. Spotted fever group rickettsiae (SFGR) has many members, and the diseases it causes include Rocky Mountain spotted fever, northern Asian fever, Queensland spotted fever, etc. The clinical manifestations are diverse, including fever, headache, rash, eschar, etc., which are similar to symptoms of other diseases (such as Lyme disease, Q fever, etc.).

[0003] More than 10 spotted fever group rickettsiae that are pathogenic to humans have been confirmed, such as the most toxic Rickettsia rickettsii and a number of newly discovered pathogens such as Rickettsia japonica, Rickettsia sibirica, Rickettsia mites, and Rickettsia conorii.

[0004] Rickettsia sibirica, also known as North Asian tick-borne Rickettsia, is an obligate intracellular parasite that can cause North Asian tick-borne spotted fever (or North Asian ticktyphus), with clinical manifestations including fever, eschar, macules, etc., accompanied by swollen lymph nodes. It is mainly transmitted to humans through tick bites, and small wild animals such as voles, squirrels, weasels and hares are naturally infected animal reservoir hosts.

[0005] Rickettsia rickettsii is an obligate intracellular parasite, a Gram-negative purple bacterium belonging to the class of α-Proteobacteria. It is aerobic and sensitive to antibiotics. It is the pathogen that causes Rocky Mountains potted fever (RMSF). Clinical manifestations include fever, headache, rash, swollen lymph nodes, and muscle and joint pain. It is mainly transmitted to humans through tick bites. Timely antibiotic treatment is crucial to reduce the mortality rate, which can be as high as 20% to 30% without treatment.

[0006] Rickettsia gracilis is the pathogen that causes Rickettsial pox and is transmitted to humans primarily through mite bites. The primary vector of transmission is the midge mite, especially the larval stage. Patients infected with R. gracilis usually present with focal skin lesions and a systemic vesicular papule rash. A small button-like ulcer covered with a black crust (eschar) appears at the site of the bite, and about a week later, fever with chills and sweats, headache, discomfort when the eyes are exposed to light, muscle pain, and a widespread rash develop. Nearby lymph nodes may swell.

[0007] Rickettsia conorii is the pathogen that causes Mediterranean spotted fever, which can cause fever, rash and flu-like symptoms. It is mainly transmitted by ticks and is also a highly pathogenic rickettsia.

[0008] Rickettsia japonicum is the pathogen that causes Japanese spotted fever (JSF), which is mainly transmitted to humans through tick bites. After sucking the blood of animals infected with Rickettsia japonicum, ticks become carriers and then spread the pathogen by biting humans. Its typical clinical symptoms include fever, headache, rash, and eschar. Severe patients may develop serious complications such as sepsis and multiple organ failure, or even die. Because Japanese rickettsial disease is uncommon, it is very easy to miss or misdiagnose, and patients will rapidly deteriorate or even die after developing severe symptoms.

[0009] Rickettsial pathogens have many members, complex antigenic structures, and obvious interspecies crossover, making it difficult to identify them. In addition, the symptoms of diseases caused by rickettsial pathogens are relatively similar, and treatment usually requires specific antibiotics, otherwise the development of severe illness may endanger the patient's life. Therefore, identifying them can help distinguish these diseases, avoid misdiagnosis, ensure that patients receive timely and correct treatment, and reduce the risk of the disease progressing to severe illness; in addition, rickettsial diseases are a type of natural zoonotic disease that seriously threatens human health. Differential diagnosis helps collect epidemiological information and understand the geographical distribution and epidemic trends of different rickettsial diseases, which is crucial for protecting public health security and formulating public health strategies and preventive measures.

[0010] Currently, common detection methods for Rickettsia include serological diagnosis and etiological detection. However, etiological detection requires the isolation and cultivation of Rickettsia, which is time-consuming, costly, and requires high standards of testing personnel. It is easily interfered by various factors, making the determination of test results more complicated. Serological testing diagnoses by detecting antibodies in the patient's serum. This method has poor specificity and is limited by the titer of the patient's serum antibodies. It cannot meet the needs of early diagnosis. In addition, due to the extensive antigenic cross-talk between Rickettsia, serological testing can only be used for group diagnosis.

[0011] With the development of molecular biology technology, there have been reports on the use of conventional PCR and fluorescent PCR to detect Rickettsia, but there are few reports on the simultaneous detection and identification of multiple Rickettsial pathogens.

[0012] Therefore, there is an urgent need in the art for a product that can simply, quickly and accurately detect and identify Rickettsial pathogens, shorten the time for determining the type of Rickettsial pathogens, and provide suggestions for different response measures. Summary of the invention

[0013] In view of this, in a first aspect, the present invention provides a composition for detecting and identifying Rickettsia pathogens, comprising at least four of the primer probe sets for detecting the following pathogens:

[0014] An upstream primer, a downstream primer and a probe for detecting Rickettsia sibirica as shown in SEQ ID NOs: 1, 2 and 3;

[0015] An upstream primer, a downstream primer and a probe for detecting Rickettsia rickettsii as shown in SEQ ID NOs: 4, 5 and 6;

[0016] Upstream primers, downstream primers and probes for detecting Rickettsia gracilis as shown in SEQ ID NOs: 7, 8 and 9;

[0017] The upstream primer, downstream primer and probe for detecting Rickettsia conorii as shown in SEQ ID NOs: 10, 11 and 12; or

[0018] The upstream primers, downstream primers and probe for detecting Rickettsia japonicum are shown in SEQ ID NOs: 13, 14 and 15.

[0019] The present invention adopts multiplex PCR technology, designs primer probes for the conserved sequences of various pathogens, and ensures that there is no mutual interference between the primer probes. One tube of reagent can detect at least four targets at the same time, which greatly improves the detection efficiency and provides more target detection information for small and precious nucleic acid samples.

[0020] In addition, the present invention combines PCR sensitivity with probe specificity, which greatly changes the defects of traditional PCR, shortens the reaction time, simplifies the operation steps, and enables the entire process of PCR amplification and product analysis to be carried out under closed conditions, thus avoiding false negatives and environmental contamination caused by cross-samples.

[0021] The design of the present invention has the advantages of specificity, sensitivity, rapidity, simplicity, low invasiveness and low cost, and is conducive to solving the technical difficulties in the current early screening, diagnosis and epidemiological investigation of Rickettsia pathogens.

[0022] Furthermore, the present invention provides a composition for detecting and identifying Rickettsia pathogens, comprising a primer probe set for detecting the following pathogens:

[0023] An upstream primer, a downstream primer and a probe for detecting Rickettsia sibirica as shown in SEQ ID NOs: 1, 2 and 3;

[0024] An upstream primer, a downstream primer and a probe for detecting Rickettsia rickettsii as shown in SEQ ID NOs: 4, 5 and 6;

[0025] Upstream primers, downstream primers and probes for detecting Rickettsia gracilis as shown in SEQ ID NOs: 7, 8 and 9;

[0026] An upstream primer, a downstream primer and a probe for detecting Rickettsia conorii as shown in SEQ ID NOs: 10, 11 and 12; and

[0027] The upstream primers, downstream primers and probe for detecting Rickettsia japonicum are shown in SEQ ID NOs: 13, 14 and 15.

[0028] Furthermore, the composition of the present invention also includes a primer probe set as shown below:

[0029] Detect the upstream primer, downstream primer and probe of the internal standard.

[0030] Furthermore, the fluorescent groups of the probes in each primer probe set in the composition of the present invention are different from each other and do not interfere with each other.

[0031] In this article, "different from each other and not interfering with each other" means that the fluorescent groups used in each probe in the composition are different and will not affect each other's detection, that is, different channels can be used for detection. For example, QUASAR705, FAM, HEX, ROX and CY5 can be used. The absorbance values ​​of these groups are not close, and different channels can be selected, so they will not interfere with each other.

[0032] In some specific embodiments, the fluorescent reporter group of the probe for Rickettsia sibirica is QUASAR705; the fluorescent reporter group of the probe for Rickettsia rickettsii is FAM; the fluorescent reporter group of the probe for Rickettsia tachinensis is ROX; the fluorescent reporter group of the probe for Rickettsia conorii is HEX; and the fluorescent reporter group of the probe for Rickettsia japonicus is CY5.

[0033] Furthermore, when the QUASAR705 channel test is positive, the sample is infected with Rickettsia sibirica.

[0034] Furthermore, when the FAM channel test is positive, the sample is infected with Rickettsia rickettsii.

[0035] Furthermore, when the ROX channel test is positive, the sample is infected with Rickettsia mitochondriasis.

[0036] Furthermore, when the HEX channel test is positive, the sample is infected with Rickettsia conorii.

[0037] Furthermore, when the CY5 channel test is positive, the sample is infected with Rickettsia japonicum.

[0038] The term "positive detection" means that the fluorescence channel corresponding to the probe in each nucleic acid composition has an obvious S-shaped amplification curve, and the Ct value is ≤40.

[0039] Further, in some embodiments, the composition of the present invention may include one or more of the above primer and probe pairs. In the present invention, "set" refers to mutually matching upstream primers, downstream primers and probes for detecting a target.

[0040] The composition of the present invention can be combined into any combination of the corresponding five targets. Those skilled in the art can combine as needed to detect which targets, that is, to combine the primers and probe pairs corresponding to the targets. These combinations are all included in the present invention.

[0041] For example, it may include any 4 of the above 5 sets of primers and probes, it may include any 3 of the above 5 sets of primers and probes, it may include any 2 of the above 5 sets of primers and probes, or it may include any 1 of the above 5 sets of primers and probes.

[0042] In some specific embodiments, the compositions of the present invention are used for fluorescent PCR.

[0043] Furthermore, the 3' end of the probe also has a non-fluorescent quencher.

[0044] Furthermore, the 3' end of the probe also has a quencher group, such as BHQ1 or BHQ2.

[0045] In a specific embodiment, the quencher group at the 3' end of the probe is BHQ1.

[0046] Furthermore, the components of the composition of the present invention may be present in a mixed form.

[0047] In a second aspect, the present invention provides use of the above-mentioned composition of the present invention in preparing a kit for detecting and identifying Rickettsia pathogens.

[0048] In a third aspect, the present invention provides a kit for detecting and identifying Rickettsia pathogens, wherein the kit comprises the composition of the present invention as described above.

[0049] Furthermore, the kit also includes a negative quality control product and a positive quality control product.

[0050] In a specific embodiment, the negative control is at least one of DEPC H2O and physiological saline. The positive control is at least one of the plasmids or fragment genes of the corresponding targets such as Rickettsia sibirica, Rickettsia rickettsii, Rickettsia spiderii, Rickettsia conorii and Rickettsia japonicus.

[0051] Furthermore, the kit also includes dNTP(U)s, PCR buffer and Mg 2+ At least one of .

[0052] Furthermore, the kit also includes: at least one of a nucleic acid releasing reagent, a nucleic acid extracting reagent, and a nucleic acid amplification reagent.

[0053] Furthermore, the kit also includes a nucleic acid release reagent, a nucleic acid extraction reagent, dNTP(U)s, a DNA polymerase, a UNG enzyme, a PCR buffer, and Mg 2+ At least one of .

[0054] Further, the concentration of the UNG enzyme is 1U / reaction to 5U / reaction. Further, the concentration of the H-Taq enzyme is 5U / reaction to 15U / reaction.

[0055] In a specific embodiment, the kit of the present invention comprises UNG enzyme, H-Taq enzyme, MgCl2, dNTP(U)s, primers, probes and PCR buffer.

[0056] PCR buffer components include Tris-HCl, NaCl, NH4Cl, and Triton X-100.

[0057] Furthermore, the total volume in a single PCR reaction tube is 20 μL to 50 μL.

[0058] In the present invention, the use of a small-volume reaction system can reduce the amount of reagents required, thereby reducing experimental costs; the small-volume system can accelerate the rate of temperature change during the PCR reaction; the use of a small-volume reaction system can reduce cross-contamination between samples, which is more important in high-sensitivity detection; when the amount of template DNA is limited, the small-volume system can reduce sample loss and ensure that there is enough template for effective amplification.

[0059] In a fourth aspect, a method for detecting and identifying Rickettsia pathogens for non-diagnostic purposes is provided, the method comprising the following steps:

[0060] 1) Extracting or releasing nucleic acid from the sample to be tested;

[0061] 2) using the composition of the present invention or the kit of the present invention as described above to perform real-time fluorescence quantitative PCR on the nucleic acid obtained in step 1);

[0062] 3) Obtain and analyze the results.

[0063] In the present invention, the sample used for detection may be blood, urine, feces, etc., but is not limited thereto.

[0064] In a specific embodiment, a composition is provided for preparing a reagent for detecting and identifying Rickettsia pathogens, wherein the detection comprises the following steps:

[0065] 1) Extracting nucleic acid from the sample to be tested;

[0066] 2) using the composition of the present invention or the kit of the present invention as described above to perform real-time fluorescence quantitative PCR on the nucleic acid obtained in step 1);

[0067] 3) Obtain and analyze the results.

[0068] In this context, the term "non-diagnostic purpose" refers to the purpose of not obtaining information on whether an individual is infected with the above pathogens and suffers from rickettsial infection. For example, it may be desirable to detect the presence of the above pathogens in a test culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 The result graph of the detection of the mixed sample of Rickettsia rickettsii, Rickettsia conorii, Rickettsia spiderii, Rickettsia japonicum and Rickettsia sibirica for the composition of the present invention.

[0070] Figure 2 The result graph of the detection of a mixed sample of Rickettsia rickettsii, Rickettsia conorii and Rickettsia spiderii for the composition of the present invention.

[0071] Figure 3 The result graph of the detection of a mixed sample of Rickettsia rickettsii, Rickettsia conorii, Rickettsia spiderii and Rickettsia sibirica for the composition of the present invention.

[0072] Figure 4 A graph showing the results of testing a mixed sample of Rickettsia rickettsii, Rickettsia conorii, Rickettsia japonicum and Rickettsia sibirica for the composition of the present invention.

[0073] Figure 5 This is a graph showing the results of the composition of the present invention detecting 500 copies / mL of Rickettsia rickettsii.

[0074] Figure 6 This is a graph showing the results of the composition of the present invention detecting 500 copies / mL of Rickettsia conorii.

[0075] Figure 7 This is a graph showing the results of the composition of the present invention detecting Rickettsia scoparia at 500 copies / mL.

[0076] Figure 8 This is a graph showing the results of the composition of the present invention detecting 500 copies / mL of Rickettsia japonicum.

[0077] Fig. 9 This is a graph showing the results of the composition of the present invention detecting 500 copies / mL of Rickettsia sibirica.

[0078] Fig.10 This is a graph showing the specificity results of the primer probe set for detecting Rickettsia sibirica of the present invention.

[0079] Fig.11 It is a specific result diagram of the composition of the present invention.

[0080] Fig.12 It is the test result figure of the comparative example composition. DETAILED DESCRIPTION

[0081] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are used to illustrate the present invention, rather than to limit the present invention.

[0082] Example 1: Primers and probes used in the present invention

[0083] The primers and probes used in the present invention are shown in Table 1 below:

[0084] Table 1

[0085]

[0086] Among them, the fluorescent reporter group of the probe of Rickettsia sibirica is QUASAR705; the fluorescent reporter group of the probe of Rickettsia rickettsii is FAM; the fluorescent reporter group of the probe of Rickettsia tachinensis is ROX; the fluorescent reporter group of the probe of Rickettsia conorii is HEX; and the fluorescent reporter group of the probe of Rickettsia japonicus is CY5.

[0087] Example 2: Method for detecting pathogens

[0088] Reagent preparation:

[0089] According to the number of samples to be tested, positive control (plasmid of the corresponding target), and negative control (normal saline), take the corresponding amount of PCR reaction solution and enzyme mixture (PCR reaction solution 19 μL / person + enzyme mixture 1 μL / person), mix them thoroughly to form a PCR mixture, centrifuge at 2000 rpm for 10 seconds and set aside.

[0090] Preparation of enzyme mixture:

[0091] The enzyme mixture is composed of a mixture of UNG enzyme and H-Taq enzyme. H-Taq enzyme (15U / μL) and UNG enzyme (5U / μL) are mixed in a certain ratio (0.75μL H-Taq enzyme and 0.25μL UNG enzyme per person).

[0092] Sample handling and loading

[0093] Take 200 μL of the sample to be tested, negative control, and positive control into a 1.5 mL centrifuge tube, and use the nucleic acid extraction or purification reagent of Shengxiang Biotechnology Co., Ltd. according to its instructions to extract nucleic acid.

[0094] 5 μL of the above-treated samples, negative controls and positive controls were respectively added to the corresponding 0.2 mL PCR reaction tubes, and 20 μL of PCR mixed solution was added to each reaction tube, and the tubes were capped.

[0095] The real-time fluorescence PCR reaction system is configured according to Table 2 below:

[0096] Table 2

[0097] Reagents Dosage PCR buffer 15.8μL <![CDATA[1M MgCl2]]> 0.2μL 100mM dNTP(U)s 0.5μL Enzyme mixture 1μL Primer SEQ ID NO: 1-2, 4-5, 7-8, 10-11, 13-14 (50 μM) 0.2μL / strip Probe SEQ ID NO: 3, 6, 9, 12, 15 (50 μM) 0.1μL / strip template 5μL

[0098] On the SLAN-96P fully automatic medical PCR analysis system, PCR amplification was performed according to a certain temperature and time setting program. The PCR amplification program was set as shown in Table 3 below:

[0099] Table 3

[0100]

[0101]

[0102] Result analysis:

[0103] If the sample FAM channel has an obvious S-type amplification curve and the Ct value is ≤40, it is judged to be positive for Rickettsia rickettsii; if the sample HEX channel has an obvious S-type amplification curve and the Ct value is ≤40, it is judged to be positive for Rickettsia conorii; if the sample ROX channel has an obvious S-type amplification curve and the Ct value is ≤40, it is judged to be positive for Rickettsia tarantula; if the sample CY5 channel has an obvious S-type amplification curve and the Ct value is ≤40, it is judged to be positive for Rickettsia japonicus; if the sample QUASAR705 channel has an obvious S-type amplification curve and the Ct value is ≤40, it is judged to be positive for Rickettsia sibiricum; if the sample FAM, HEX, ROX, CY5 and QUASAR705 channels have no amplification curve (No Ct) or the Ct value is greater than 40, it is judged to be negative, as shown in Table 4.

[0104] Table 4

[0105]

[0106] Example 3: Test results of the test samples of the composition of the present invention

[0107] The primers and probes shown in Example 1 were used to detect samples of Rickettsia rickettsii, Rickettsia conorii, Rickettsia mitochondrialis, Rickettsia japonicum and Rickettsia sibirica according to the method of Example 2. PCR detection was performed on a Macrostone fluorescent quantitative PCR instrument. The detection results are shown in FIG. Figures 1 to 4 As shown, Figure 1 The results of the mixed sample of Rickettsia rickettsii, Rickettsia conorii, Rickettsia tarantula, Rickettsia japonicum and Rickettsia sibirica are shown in Figure 1. Figure 2 The results of the mixed sample of Rickettsia rickettsii, Rickettsia conorii and Rickettsia tarantula were as follows: Figure 3 The results of the mixed sample of Rickettsia rickettsii, Rickettsia conorii, Rickettsia spiderii and Rickettsia sibirica are shown in Figure 1. Figure 4 The results of detecting a mixed sample of Rickettsia rickettsii, Rickettsia conorii, Rickettsia japonica and Rickettsia sibirica. As can be seen from the figure, the composition of the present invention can detect and distinguish Rickettsia rickettsii, Rickettsia conorii, Rickettsia tarantula, Rickettsia japonica and Rickettsia sibirica.

[0108] Example 4: Sensitivity of the composition of the present invention

[0109] The LOD (sensitivity) test was performed on Rickettsia rickettsii, Rickettsia conorii, Rickettsia tarantula, Rickettsia japonicum and Rickettsia sibirica. The results are as follows Figures 5 to 9 , showing that the composition can still accurately detect samples as low as 500 copies / mL, indicating that the sensitivity of the composition of the present invention is 500 copies / mL.

[0110] Example 5. Specificity of the composition of the present invention

[0111] The primer probe set for detecting Siberian Rickettsia in the composition of the present invention was used to detect samples of Rickettsia rickettsii, Rickettsia conorii, Rickettsia spiderii and Rickettsia japonicum. The results are as follows: Fig.10 As shown in the figure, it is shown that the primer probe set designed to detect Siberian Rickettsia can only specifically amplify the target Siberian Rickettsia, and has no cross reaction with other targets in the composition. The composition of the present invention was used to detect samples of other similar Rickettsia (Rickettsia prowazekii, Rickettsia morozekii, Rickettsia australis, Coxiella burnetii), and the results are as follows Fig.11 As shown in Table 5, there is no cross reaction with other similar Rickettsiae. The above results show that the composition of the present invention has good specificity.

[0112] Table 5

[0113]

[0114] Example 6: Anti-interference performance of the composition of the present invention

[0115] The detection was carried out according to the method described in Example 2, except that in addition to the above-mentioned samples to be tested, common interfering substances (oxymetazoline hydrochloride, dexamethasone, cefmenoxime hydrochloride, benzocaine, zanamivir, ribavirin, azithromycin, sodium chloride, SDS, EDTA, urea, heme, purified mucin, anhydrous ethanol, mupirocin, tobramycin) were additionally added to the entire system. The test results are shown in Table 6. It can be seen from Table 6 that the common interfering substances have no effect on the detection performance of the composition of the present invention.

[0116] Table 6

[0117]

[0118]

[0119] Example 7. Stability of the composition of the present invention

[0120] The composition and kit of the present invention were tested after being stored for 12 months under actual storage conditions (-20±5°C), and the performance was stable; the results of the 37°C accelerated destruction stability experiment showed that the kit was stored in a 37°C constant temperature box for 24 hours, and the results met the quality requirements; the freeze-thaw stability test showed that different boxes of kits were frozen and thawed once at each test time point at the actual storage temperature, and were tested continuously for 6 times, and the results all met the quality requirements.

[0121] Comparative Example 1: Other primers and probes designed by the present invention that have poor effects

[0122] When multiple pathogens are detected together, there are many primers and probes. Dimers are likely to occur between primers and primers, probes and probes, or primers and probes. In order to ensure the conservatism of the design (conservatism is crucial to the accuracy of the detection) and to consider the mutual interference between different primers and probes, the primers and probes need to be carefully designed.

[0123] The remaining primers and probes designed by the present invention that were not effective were used to perform the test in the same manner as in Example 2. The results were as follows: Fig.12 As shown in the figure, it can be seen that due to the interference between the primers and probes, the target fluorescence increment is affected, and the curve shape is poor compared with the composition of the present invention, not straight enough, and does not reach the expected S shape, indicating that the amplification is seriously affected. This shows that the primers and probes designed by the present invention have better amplification effects and higher sensitivity.

Claims

1. A composition for detecting and identifying Rickettsia pathogens, comprising at least four of the primer probe sets for detecting the pathogens described below: An upstream primer, a downstream primer and a probe for detecting Rickettsia sibirica as shown in SEQ ID NOs: 1, 2 and 3; An upstream primer, a downstream primer and a probe for detecting Rickettsia rickettsii as shown in SEQ ID NOs: 4, 5 and 6; Upstream primers, downstream primers and probes for detecting Rickettsia gracilis as shown in SEQ ID NOs: 7, 8 and 9; The upstream primer, downstream primer and probe for detecting Rickettsia conorii as shown in SEQ ID NOs: 10, 11 and 12; or The upstream primers, downstream primers and probe for detecting Rickettsia japonicum are shown in SEQ ID NOs: 13, 14 and 15.

2. The composition according to claim 1, characterized in that The composition includes a primer probe set for detecting the pathogens described below: An upstream primer, a downstream primer and a probe for detecting Rickettsia sibirica as shown in SEQ ID NOs: 1, 2 and 3; An upstream primer, a downstream primer and a probe for detecting Rickettsia rickettsii as shown in SEQ ID NOs: 4, 5 and 6; Upstream primers, downstream primers and probes for detecting Rickettsia gracilis as shown in SEQ ID NOs: 7, 8 and 9; An upstream primer, a downstream primer and a probe for detecting Rickettsia conorii as shown in SEQ ID NOs: 10, 11 and 12; and The upstream primers, downstream primers and probe for detecting Rickettsia japonicum are shown in SEQ ID NOs: 13, 14 and 15.

3. The composition according to claim 2, characterized in that The composition also includes a primer probe set as shown below: Detect the upstream primer, downstream primer and probe of the internal standard.

4. The composition according to claim 3, characterized in that The fluorescent groups of the probes in each primer probe group in the composition are different from each other and do not interfere with each other.

5. The composition according to claim 4, characterized in that The fluorescent reporter group of the probe for Rickettsia sibirica is QUASAR705; the fluorescent reporter group of the probe for Rickettsia rickettsii is FAM; the fluorescent reporter group of the probe for Rickettsia tarantula is ROX; the fluorescent reporter group of the probe for Rickettsia conorii is HEX; and the fluorescent reporter group of the probe for Rickettsia japonicus is CY5.

6. The composition according to any one of claims 1 to 5, characterized in that The components of the composition are present in admixture.

7. Use of the composition according to any one of claims 1 to 6 in the preparation of a kit for detecting and identifying Rickettsia pathogens.

8. A kit for detecting and identifying Rickettsia pathogens, the kit comprising the composition according to any one of claims 1 to 6.

9. The kit according to claim 8, characterized in that The kit also includes: UNG enzyme, H-Taq enzyme, MgCl2, dNTP(U)s and PCR buffer.

10. Use of a composition for preparing a reagent for detecting and identifying Rickettsia pathogens, the detection comprising the following steps: 1) Extracting nucleic acid from the sample to be tested; 2) performing real-time fluorescence quantitative PCR on the nucleic acid obtained in step 1) using the composition according to any one of claims 1 to 6 or the kit according to any one of claims 8 to 9; 3) Obtain and analyze the results.

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