Composition for detecting oral pathogenic microorganisms, kit and application

Abstract

The invention belongs to the field of molecular biology detection, particularly relates to detection of oral pathogenic microorganisms, and more particularly relates to detection of Rosella caries, Rosella mucilaginosa and Rosella airborne. According to the combined detection composition provided by the invention, different pathogens are detected by mainly utilizing a multiple fluorescent PCR analysis method and detecting target spots on the different pathogens, so that detection and distinguishing of Rosella caries, Rosella mucilaginosa, Rosella airborne and Arcanobacterium hemolyticus are simultaneously realized in a single-tube reaction system; therefore, a targeted strategy is provided for subsequent treatment. The composition provided by the invention has the advantages of higher detection sensitivity (up to 500 copies / mL), good specificity and more accurate detection, provides a more sufficient basis for rapid diagnosis and exclusion of infection of different pathogens for clinicians, shortens the time for the clinicians to diagnose the condition of a patient, and accelerates the implementation of treatment measures on the patient.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biological detection, and specifically, relates to the detection of oral pathogenic microorganisms, and more specifically, to the detection of caries Roseburia, slimy Roseburia, air Roseburia, and hemolytic cryptobacterium. Background Art

[0002] Roseburia is a gram-positive, pleomorphic bacterium that is negative for acid-fast and weak acid-fast staining. Discovered by George and Brown in 1967, it belongs to the Micrococcaceae family and is now classified as the Actinomycetes family. There are three species of Roseburia associated with clinical infections: R. dentocariosa, R. mucilaginosa, and Rothiaaeria. These bacteria are colonizers in healthy oral cavity, but can cause pathogenicity in some unhealthy oral cavity. They may also cause bloodstream infection, endocarditis or other infections. Most of the infections caused by these bacteria are related to the use of immunosuppressants to treat underlying diseases.

[0003] Cryptobacterium haemolyticum belongs to the Actinomycetaceae family, Cryptobacterium genus, and is a facultative anaerobic bacterium. They are usually distributed in the upper respiratory tract and oral environment of humans, and can parasitize in the nasopharynx. Cryptobacterium haemolyticum has a hemolytic effect. The hemolysin it produces destroys the membranes of red blood cells and other cells, causing cell lysis. This hemolysis helps the bacteria escape the attack of the immune system. Usually, people enter the body through skin incisions, respiratory tracts or digestive tracts. When Cryptobacterium haemolyticum invades the human body, it will form colonies by attaching to the surface of human cells, and then release hemolysin to destroy cells, causing infection. Common diseases include pharyngitis, otitis media, cellulitis, endocarditis, pneumonia, sepsis, etc.

[0004] Roseburia dentata, Roseburia slime, Roseburia aerogenes, and Cryptobacterium haemolyticum can all cause human bloodstream infections. At the same time, conventional identification methods for the above-mentioned species have poor specificity and are easily mistaken for other species. Therefore, there is a need in the art for a product that can simply and quickly detect the above-mentioned pathogens, and has good sensitivity and specificity, and can detect and distinguish the above-mentioned pathogens that cause infection, so as to provide targeted strategies for the prevention and treatment of the above-mentioned infections. Summary of the invention

[0005] In view of this, in a first aspect, the present invention provides a composition for detecting oral pathogenic microorganisms, comprising:

[0006] Upstream primers, downstream primers and probes for detecting Roseburia dentata as shown in SEQ ID NOs: 1 to 3;

[0007] Upstream primers, downstream primers and probes for detecting Roseburia muciniphila as shown in SEQ ID NOs: 4 to 6; and

[0008] The upstream primers, downstream primers and probes for detecting Roseburia aeruginosa are shown in SEQ ID NOs: 7-9.

[0009] The combined detection composition provided by the present invention mainly utilizes a multiplex fluorescent PCR analysis method to detect different pathogens by detecting targets on different pathogens, thereby simultaneously realizing the detection and differentiation of caries Roseburia, slimy Roseburia, and air Roseburia in a single-tube reaction system, so as to provide a targeted strategy for subsequent treatment. The composition of the present invention has a higher detection sensitivity of 500 copies / mL, good specificity, and more accurate detection, providing clinicians with a relatively sufficient basis for rapid diagnosis and exclusion of different pathogen infections, shortening the time for clinicians to diagnose the patient's condition, and accelerating the implementation of treatment measures for the patient.

[0010] Furthermore, the composition further comprises:

[0011] The upstream primers, downstream primers and probe for detecting Cryptobacillinamyces hemolyticus are shown in SEQ ID NOs: 10 to 12.

[0012] Furthermore, the composition includes an upstream primer, a downstream primer and a probe for detecting an internal standard.

[0013] In some specific embodiments, an exogenous internal standard is added to the sample, and further, the internal standard is a human internal standard gene.

[0014] In a specific embodiment, the internal standard is RNase P or NADPH.

[0015] Furthermore, the fluorescent groups of the probes used in the composition of the present invention to detect different pathogens are different from each other and do not interfere with each other.

[0016] 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, ATTO425, 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.

[0017] In some specific embodiments, the fluorescent reporter group of the probe for detecting caries Roseburia is FAM; the fluorescent reporter group of the probe for detecting slime-slippery Roseburia is HEX (or VIC); the fluorescent reporter group of the probe for detecting air Roseburia is ROX; and the fluorescent reporter group of the probe for detecting Cryptobacterium haemolyticum is CY5.

[0018] 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 upstream and downstream primers and probes that match each other for detecting a target.

[0019] The composition of the present invention can be arbitrarily combined into any combination form for detecting the corresponding four 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. The technical effect of the combination that can be successfully detected is also expected by those skilled in the art.

[0020] For example, any three of the four sets of primers and probes may be included, any two of the four sets of primers and probes may be included, or any one of the four sets of primers and probes may be included.

[0021] In some specific embodiments, the compositions of the present invention are used for fluorescent PCR.

[0022] Furthermore, the 3' end of the probe also has a non-fluorescent quencher.

[0023] Furthermore, the 3' end of the probe also has a quencher group, such as BHQ1 or BHQ2.

[0024] In a specific embodiment, the 3' end of the probe is BHQ1.

[0025] In a specific embodiment, each component of the composition of the present invention is present in a separate package.

[0026] In a particular embodiment, the components of the composition of the present invention are present in the same package.

[0027] Furthermore, the components of the composition of the present invention are present in a mixed form.

[0028] In a second aspect, the present invention provides a use of the above-mentioned composition of the present invention in preparing a kit for detecting oral pathogenic microorganisms, wherein the pathogen is one or more of Roseburia dentata, Roseburia slime, Cryptobacillaceae haemolytica, or Roseburia aeruginosa.

[0029] In a third aspect, the present invention provides a kit for detecting oral pathogenic microorganisms, wherein the kit comprises the composition of the present invention as described above.

[0030] Furthermore, the kit also includes a negative quality control product and a positive quality control product.

[0031] In a specific embodiment, the negative control is at least one of DEPC H2O and normal saline; the positive control is at least one of the fragment plasmid or positive strain of Roseburia dentata, Roseburia mucosae, Roseburia aerogenes, and Cryptobacterium haemolyticum.

[0032] Furthermore, the kit also includes dNTP(U)s, PCR buffer and Mg 2+ At least one of .

[0033] Furthermore, the kit also includes at least one of DNA polymerase and uracil glycosylase (UNG).

[0034] Furthermore, the kit also includes at least one of a nucleic acid releasing reagent and a nucleic acid extracting reagent.

[0035] Furthermore, the kit also includes a nucleic acid release reagent, a nucleic acid extraction reagent, dNTP, dUTP, uracil glycosylase (UNG), DNA polymerase, PCR buffer and Mg 2+ At least one of .

[0036] Furthermore, the concentration of the DNA polymerase is 5 U / reaction to 15 U / reaction, for example, the DNA polymerase may be Taq enzyme.

[0037] In a specific embodiment, the kit of the present invention comprises Taq enzyme, UDG enzyme, Mg 2+ , dNTP(U)s, primers, probes and PCR buffer.

[0038] The main components of PCR buffer include Tris-HCl, NaCl, NH4Cl, and Tween 20.

[0039] In some specific embodiments, the total volume of the reactants in a single PCR reaction tube is 20 μL to 60 μL.

[0040] In a specific embodiment, the kit of the present invention is compatible with a digital PCR amplification system, that is, it can be directly used for amplification on a digital PCR instrument.

[0041] In a fourth aspect, a method for detecting oral pathogenic microorganisms is provided, the method comprising the following steps:

[0042] 1) Extracting nucleic acid from the sample to be tested;

[0043] 2) using the composition of the present invention or the kit of the present invention as described above to perform fluorescent PCR detection on the nucleic acid obtained in step 1);

[0044] 3) Obtain and analyze the results.

[0045] In the present invention, the sample used for detection may be blood, saliva, etc., but is not limited thereto.

[0046] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0047] UNG enzyme reaction, temperature is 50-60℃, time is 0.5-6min, 1 cycle; Taq enzyme activation, temperature is 90-95℃, time is 0.5-6min, 1 cycle; denaturation, temperature is 90-95℃, time is 5-20 seconds, annealing, temperature is 55℃-60℃, time is 10-60 seconds, 30-50 cycles, and fluorescence is collected.

[0048] Furthermore, the method for detecting oral pathogenic microorganisms is for non-diagnostic purposes.

[0049] In a specific embodiment, a composition is provided for preparing a reagent for detecting oral pathogenic microorganisms, wherein the detection comprises the following steps:

[0050] 1) Extracting nucleic acid from the sample to be tested;

[0051] 2) using the composition of the present invention or the kit of the present invention as described above to perform fluorescent PCR detection on the nucleic acid obtained in step 1);

[0052] 3) Obtain and analyze the results.

[0053] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0054] UNG enzyme reaction, temperature is 50-60℃, time is 0.5-6min, 1 cycle; Taq enzyme activation, temperature is 90-95℃, time is 0.5-6min, 1 cycle; denaturation, temperature is 90-95℃, time is 5-20 seconds, annealing, temperature is 55℃-60℃, time is 10-60 seconds, 30-50 cycles, and fluorescence is collected.

[0055] In this context, the term "non-diagnostic purpose" means that the method is not intended to obtain information on whether an individual is infected with the above pathogens and suffers from the infection. For example, the method can detect whether the environment, culture, and drinking water sources have the above pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 This is a graph showing the test results of the composition of the present invention (Roxella dentata, Roxella slime, Roxella aerogenes, and Cryptobacterium hemolyticum);

[0057] Figures 2 to 5 is a sensitivity result diagram of the composition of the present invention (Roxella dentata, Roxella slime, Roxella aerogenes, and Cryptobacterium hemolyticum, respectively);

[0058] Figure 6 It is a specific result diagram of the composition of the present invention;

[0059] Figures 7-10 The results of the anti-interference of the composition of the present invention are shown in Figure (Roxella dentata, Roxella sluggella, Roxella aerogenes, and Cryptobacterium hemolyticum);

[0060] Figures 11 to 14 The accelerated stability result diagram of the composition of the present invention (Roxella dentata, Roxella slime, Roxella aerogenes, and Cryptobacterium hemolyticum, respectively);

[0061] Fig.15 This is a graph showing the test results of the comparative example composition of the present invention. DETAILED DESCRIPTION

[0062] 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.

[0063] Example 1: Primers and probes used in the present invention

[0064] The primers and probes used in the present invention are shown in Table 1 below:

[0065] Table 1

[0066]

[0067] Among them, the fluorescent reporter group of the caries Roseburia probe is FAM; the fluorescent reporter group of the slimy Roseburia probe is HEX; the fluorescent reporter group of the air Roseburia is ROX; and the fluorescent reporter group of the hemolytic Cryptobacillus probe is CY5.

[0068] Example 2: Method for detecting pathogens

[0069] Reagent preparation:

[0070] Preparation of enzyme mixture:

[0071] The DNA polymerase mixture is composed of UNG and H-Taq enzymes, which are prepared by mixing H-Taq enzyme (5U / μL) and UNG enzyme (2U / μL) in a certain ratio.

[0072] According to the number of samples to be tested, positive controls, and negative controls, take the corresponding amount of PCR reaction solution and enzyme mixture (PCR reaction solution 43 μL / person + enzyme mixture 2 μL / person), mix them thoroughly to form a PCR mixture, centrifuge at 2000 rpm for 10 s and set aside. The real-time fluorescence PCR reaction system is configured according to the following Table 2.

[0073] Table 2

[0074] Components Volume / concentration in each reaction PCR Buffer 39.75μL UNG enzyme (2U / μL) 0.05μL Taq enzyme (5U / μL) 1.95μL <![CDATA[Mg 2+ ]]> 0.25μL(1M) dNTP 1μL Primer (50pmol / μL) 0.2μL / strip Probe (50pmol / μL) 0.1μL / strip

[0075] Sample handling and loading:

[0076] The test sample of the present invention is blood. The magnetic bead method is used to extract viral nucleic acid. The kits used are all commercially available kits of Shengxiang Biotechnology. The following operations are performed in the sample processing room:

[0077] Take a 1.5mL cryotube and add glass beads. Add 300μL of the treated sample and 300μL of TE to the 1.5mL cryotube containing the mixed glass beads and grind it using a grinder. After the grinding is completed, take out the cryotube and centrifuge it instantly. Take out the supernatant liquid as the sample to be tested for later use.

[0078] Take a number of 1.5mL centrifuge tubes according to the number of samples to be tested, and add 300μL of the sample to be tested to each tube;

[0079] Add 500 μL of extraction solution 1 and 50 μL of proteinase K-magnetic bead mixture; cover the tube, shake and mix for 30 seconds, and heat at 60°C for 10 minutes.

[0080] Let stand at room temperature for 1 min, centrifuge at low speed, place the centrifuge tube on the magnetic separator, and slowly aspirate and discard the waste liquid after 5 min (*Be careful not to touch the magnetic beads adsorbed on the inner side of the tube wall);

[0081] Add 200 μL of washing solution 1 and 600 μL of washing solution 2, shake and mix for 30 seconds, centrifuge at low speed, and place the centrifuge tube in a magnetic separator. Magnetic suction for 3 minutes, completely aspirate and discard the liquid.

[0082] Place the centrifuge tube in a centrifuge and centrifuge it at low speed for a short time, then place the centrifuge tube in a magnetic separator again. Magnetic suction for 3 minutes to completely absorb the liquid at the bottom of the tube.

[0083] Add 30-100 μL of elution buffer S (80 μL is recommended), shake and mix for 30 seconds to elute the magnetic beads on the wall of the centrifuge tube to the bottom of the tube, and let it stand at room temperature for 3 minutes; centrifuge at a low speed, place the centrifuge tube on the magnetic separator again for 3 minutes, and then transfer the eluted nucleic acid to a clean 1.5 mL centrifuge tube.

[0084] 5 μL of the above-treated samples, positive controls and negative controls were respectively added to the corresponding 0.2 mL PCR reaction tubes, 45 μL of PCR mixture was added to each tube, and the tubes were capped.

[0085] PCR amplification:

[0086] PCR amplification was performed on a SLAN-96P real-time fluorescence quantitative PCR instrument according to the temperature and time settings shown in Table 3.

[0087] Table 3

[0088]

[0089] Result analysis and judgment

[0090] After the reaction is completed, the results are automatically saved, and the amplification curves of the detection targets are analyzed separately. According to the analyzed image, adjust the Start value, End value, and Threshold value of the Baseline (users can adjust according to actual conditions, the Start value can be set between 3 and 15, and the End value can be set between 5 and 20, and adjust the amplification curve of the negative control to make it flat or below the threshold line), click Analyze to analyze, so that each parameter meets the requirements of the following "Quality Control", and then record the qualitative results in the Plate window.

[0091] Quality Control:

[0092] If the sample has an obvious S-shaped amplification curve in the FAM, HEX (VIC), ROX, CY5 channels and the Ct value is ≤40, it is judged as positive; if the sample has no amplification curve (No Ct) or the Ct value is greater than 40 in the FAM, HEX (VIC), ROX, CY5 channels, it is judged as negative. The details are shown in Table 4 below.

[0093] Table 4

[0094]

[0095] Example 3: Test results of the test samples of the composition of the present invention

[0096] The primers and probes shown in Example 1 were used to perform PCR detection on a mixed sample of caries Roseburia, slimy Roseburia, aerobic Roseburia, and hemolytic Arcanobacterium using a SLAN-96P real-time fluorescence quantitative PCR instrument according to the method of Example 2. The detection results are shown in FIG. Figure 1 As shown in the figure, it can be seen that the composition of the present invention can perform good detection on the mixed sample of the four pathogens of the present invention.

[0097] Example 4: Sensitivity of the composition of the present invention

[0098] Using the composition in Example 1 of the present invention, a gradient dilution of the plasmid was taken, with concentrations of 2000, 1000, 500, and 100 copies / mL, respectively. The four concentrations were tested, and 5 μL was taken as a template. The results are as follows: Figures 2 to 5As shown, it is shown that each channel can still accurately detect samples as low as 500 copies / mL, with a detection rate of 100%, indicating that the sensitivity of the composition of the present invention is 500 copies / mL.

[0099] Example 5. Specificity of the composition of the present invention

[0100] The composition of the present invention has no cross reaction with other pathogens (Klebsiella pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, Legionella pneumophila, Bordetella pertussis, Staphylococcus aureus, Mycoplasma pneumoniae, Chlamydia pneumoniae, Mycobacterium tuberculosis, nontuberculous mycobacteria, Acinetobacter baumannii, Acinetobacter junii, Rhodococcus equi, Candida albicans, Candida tropicalis, Candida krusei). The results are as follows Figure 6 As shown, it is shown that the composition of the present invention has good specificity.

[0101] Example 6: Anti-interference and stability of the composition of the present invention

[0102] The composition of Example 1 was analyzed for the addition of interfering substances. The experimental results showed that potential PCR inhibitors / interfering substances such as 10 μg / mL mucin, 20 μg / mL heme, 0.44 mg / mL sulfamethoxazole, 0.8 mg / mL amphotericin B, 0.5 mg / mL penicillin, 0.2 mg / ml oxacillin, 0.1 mg / ml gentamicin, 0.2 mg / mL clindamycin, 0.1 g / ml azithromycin, 0.5 mg / mL ciprofloxacin, and 0.5 mg / mL vancomycin had no significant effect on the kit. The results are shown in Figures 7 to 10. The results of the accelerated stability test are shown in Figures 7 to 10. Figures 11 to 14 As shown in the figure, it can be seen that the sensitivity of the test after acceleration is not affected, indicating that the composition of the present invention has good stability.

[0103] Comparative Example 1: Other primers and probes designed by the present invention that have poor effects

[0104] Due to the principle of complementary base pairing, dimers will form between primers and (or) probes, but this probability is very small and can be ruled out at the beginning of the design. However, when multiple pathogens are detected together, there are many primers and probes, and 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.

[0105] Therefore, the inventors also designed some other primers and probes to form different detection systems (sequences not shown), which are also used to detect the above pathogens. Fig.15As shown in the figure, it can be seen that the results show that only part of the amplification curve appears in the detection, and the amplification curve has a low increase and poor repeatability, while other targets do not even have amplification curves. Therefore, the overall detection effect is poor.

Claims

1. A composition for detecting oral pathogenic microorganisms, comprising: Upstream primers, downstream primers and probes for detecting Roseburia dentata as shown in SEQ ID NOs: 1 to 3; Upstream primers, downstream primers and probes for detecting Roseburia muciniphila as shown in SEQ ID NOs: 4 to 6; and The upstream primers, downstream primers and probes for detecting Roseburia aeruginosa are shown in SEQ ID NOs: 7-9.

2. The composition according to claim 1, characterized in that The composition further comprises: The upstream primers, downstream primers and probe for detecting Cryptobacillinamyces hemolyticus are shown in SEQ ID NOs: 10 to 12.

3. The composition according to claim 2, characterized in that The fluorescent reporter group of the probe for detecting caries Roseburia is FAM; the fluorescent reporter group of the probe for detecting slimy Roseburia is HEX or VIC; the fluorescent reporter group of the probe for detecting air Roseburia is ROX; and the fluorescent reporter group of the probe for detecting hemolytic Cryptobacillus is CY5.

4. The composition according to any one of claims 1 to 3, characterized in that The composition also includes an upstream primer, a downstream primer and a probe for detecting an internal standard.

5. The composition according to claim 4, characterized in that The components of the composition are present in admixture.

6. Use of the composition according to any one of claims 1 to 5 in preparing a kit for detecting oral pathogenic microorganisms, wherein The pathogen is one or more of Cryptobacterium haemolyticum, Roseburia dentata, Roseburia mucosae, or Roseburia aeruginosa.

7. A kit for detecting oral pathogenic microorganisms, comprising the composition according to any one of claims 1 to 5.

8. The kit according to claim 7, characterized in that The kit also includes a negative quality control product and a positive quality control product.

9. The kit according to claim 7 or 8, characterized in that The kit also includes: a nucleic acid release reagent, a nucleic acid extraction reagent, a DNA polymerase, dNTP, dUTP, UNG enzyme, a PCR buffer, or Mg 2+ At least one of .

10. Use of a composition for preparing a reagent for detecting oral pathogenic microorganisms, the detection comprising the following steps: 1) Extracting nucleic acid from the sample to be tested; 2) performing fluorescent PCR detection on the nucleic acid obtained in step 1) using the composition according to any one of claims 1 to 5 or the kit according to any one of claims 7 to 9; 3) Obtain and analyze the results.

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