Composition for detecting wound infection related pathogens, kit and application

Abstract

The invention belongs to the field of molecular biology detection, particularly relates to detection of pathogens related to wound infection, and more particularly relates to detection of at least four of Vinci, Corynebacterium striatum, anaerobic peptococcus, non-glycolytic peptococcus and anaerococcus przewalskii. The composition disclosed by the invention is higher in detection sensitivity (up to 500 copies / mL), good in specificity and more accurate in detection, and can provide a relatively sufficient basis for rapid detection and elimination of infection of different pathogens, shorten the judgment time of wound infection inducements and accelerate implementation of countermeasures.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biological detection, and specifically, relates to the detection of pathogens related to wound infection, and more specifically, to the detection of Peptostreptococcus anaerobicus, Peptostreptococcus insaccharolyticus, and Anaerobic coccus prausnitzii. Background Art

[0002] Finegoldia magna is a genus of Gram-positive, anaerobic cocci of the Clostridium class that is an opportunistic human pathogen that often colonizes the skin and mucous membranes and often occurs in biofilms of chronic ulcers, such as diabetic foot or decubitus ulcers. Finegoldia magna is a normal commensal of human skin and mucous membranes and is primarily found in the skin, gastrointestinal tract, and urogenital tract. It accounts for 5-12% of anaerobic infections and 20-40% of Gram-positive anaerobes. This pathogen has been associated with joint infections, skin and soft tissue infections after prosthetic implantation.

[0003] Bacteroides ureolyticus is a species of Gram-negative obligate anaerobic bacteria. It is a common intestinal bacterium and is usually non-pathogenic, but may cause infection when the immune function is weakened, such as abscesses in the abdominal cavity, pelvic cavity, etc.

[0004] Peptostreptococcus anaerobius, Peptoniphilus asaccharolyticus, and Anaerococcus prevotii are all members of the genus Peptostreptococcus, a genus of Gram-positive bacteria in the family Peptostreptococci of the order Eubacteriales. This genus is an obligate parasite of the oral cavity, mucous membranes, and intestines of mammals and may play a role in purulent infections. Peptostreptococci are slow-growing bacteria that are sometimes resistant to antimicrobial drugs. Under conditions of immunosuppression or trauma, these bacteria can become pathogenic and cause sepsis, damaging the host. Peptostreptococci can cause brain, liver, breast, and lung abscesses, as well as systemic necrotizing soft tissue infections.

[0005] The detection of wound infection is of great clinical significance. Timely detection and confirmation of pathogens of wound infection can help prevent the infection from spreading further in the patient's body, especially in patients with weak immune systems, such as diabetics, the elderly or postoperative patients. Early detection helps reduce the risk of infection spreading to other tissues or even the whole body. Wound infection usually involves complex microorganisms, including pathogens with strong drug resistance. Testing can provide information on the types of infectious pathogens and their drug resistance, help doctors choose the most effective antimicrobial drugs, reduce unnecessary use of antibiotics, and thus reduce the risk of antibiotic resistance. If wound infection is not effectively controlled, it may lead to local or systemic complications, such as sepsis and tissue necrosis. Rapid detection and identification of infectious pathogens can help take targeted measures in a timely manner, thereby reducing the probability of serious complications; for patients with surgical wounds, timely detection of wound infection and confirmation of the type of infectious pathogen can avoid delayed wound healing or other postoperative complications caused by postoperative infection, support the postoperative recovery process, and reduce hospitalization time.

[0006] Therefore, there is a need in the art for a product that can simply and quickly detect the above pathogens, and has good sensitivity and specificity, and can detect and distinguish pathogenic microorganisms that cause wound infections, so as to provide targeted strategies for the prevention and treatment of the above wound infections. Summary of the invention

[0007] In view of this, in a first aspect, the present invention provides a composition for detecting pathogens associated with wound infection, comprising upstream and downstream primers and probes for detecting at least 4 of the following pathogens:

[0008] Upstream primers, downstream primers and probes for detecting Bacillus thuringiensis as shown in SEQ ID NOs: 1 to 3;

[0009] Upstream primers, downstream primers and probes for detecting Corynebacterium striatum as shown in SEQ ID NOs: 4 to 6;

[0010] Upstream primers, downstream primers and probes for detecting anaerobic peptostreptococci as shown in SEQ ID NOs: 7 to 9;

[0011] The upstream primers, downstream primers and probe for detecting Peptostreptococcus insaccharolyticus as shown in SEQ ID NOs: 10 to 12; or

[0012] The upstream primers, downstream primers and probe for detecting Anaerococcus provocateurus are shown in SEQ ID NOs: 13-15.

[0013] 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 at least 4 of Fingoldella, Corynebacterium striata, Peptostreptococcus anaerobicus, Peptostreptococcus insaccharolyticus, and Anaerococcus provosa in a single-tube reaction system, so as to provide a targeted strategy for wound infection prevention and treatment. The composition of the present invention has a higher detection sensitivity of 500 copies / mL, good specificity, and more accurate detection, and can provide a relatively sufficient basis for rapid detection and exclusion of infection by different pathogens, shorten the judgment time of the cause of wound infection, and accelerate the implementation of countermeasures.

[0014] Furthermore, the present invention provides a composition for detecting pathogens related to wound infection, comprising:

[0015] Upstream primers, downstream primers and probes for detecting Bacillus thuringiensis as shown in SEQ ID NOs: 1 to 3;

[0016] Upstream primers, downstream primers and probes for detecting Corynebacterium striatum as shown in SEQ ID NOs: 4 to 6;

[0017] Upstream primers, downstream primers and probes for detecting anaerobic peptostreptococci as shown in SEQ ID NOs: 7 to 9;

[0018] Upstream primers, downstream primers and probes for detecting Peptostreptococcus insaccharolyticus as shown in SEQ ID NOs: 10 to 12; and

[0019] The upstream primers, downstream primers and probe for detecting Anaerococcus provocateurus are shown in SEQ ID NOs: 13-15.

[0020] Furthermore, the composition includes an upstream primer, a downstream primer and a probe for detecting an internal standard.

[0021] In some specific embodiments, an exogenous internal standard is added to the sample, and further, the internal standard is a human internal standard gene.

[0022] In a specific embodiment, the internal standard is RNase P or GAPDH.

[0023] 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.

[0024] 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.

[0025] In some specific embodiments, the fluorescent reporter group of the probe for detecting Bacillus thuringiensis is FAM; the fluorescent reporter group of the probe for detecting Corynebacterium striata is HEX; the fluorescent reporter group of the probe for detecting anaerobic Streptococcus is ROX; the fluorescent reporter group of the probe for detecting insuferous Streptococcus is CY5; and the fluorescent reporter group of the probe for detecting Anaerobic Coccus provocateur is QUASAR 705.

[0026] 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 primers, downstream primers and probes that match each other to detect a target.

[0027] 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 upstream and downstream primers and probe pairs corresponding to the targets. These combinations are all included in the present invention.

[0028] For example, it may include any 4 of the above 5 sets of primers and probes, any 3 of the above 5 sets of primers and probes, any 2 of the above 5 sets of primers and probes, or any 1 of the above 5 sets of primers and probes.

[0029] In some specific embodiments, the compositions of the present invention are used for fluorescent PCR.

[0030] Furthermore, the 3' end of the probe also has a non-fluorescent quencher.

[0031] Furthermore, the 3' end of the probe also has a quencher group, such as BHQ1 or BHQ2.

[0032] In a specific embodiment, the quencher group at the 3' end of the probe is BHQ1.

[0033] In a specific embodiment, each component of the composition of the present invention is present in a separate package.

[0034] In a particular embodiment, the components of the composition of the present invention are present in the same package.

[0035] Furthermore, the components of the composition of the present invention are present in a mixed form.

[0036] In a second aspect, the present invention provides the use of the above-mentioned composition of the present invention in the preparation of a kit for detecting pathogens related to wound infection, wherein the pathogen is one or more of Peptostreptococcus insaccharolyticus, Peptostreptococcus quinquefolius, Corynebacterium striata, Peptostreptococcus anaerobicus, or Anaerococcus provocateurans.

[0037] In a third aspect, the present invention provides a kit for detecting pathogens associated with wound infection, wherein the kit comprises the composition of the present invention as described above.

[0038] Furthermore, the kit also includes a negative quality control product and a positive quality control product.

[0039] 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 a fragment plasmid or a positive strain of Peptostreptococcus aglycolyticus, Peptostreptococcus magna, Corynebacterium striata, Peptostreptococcus anaerobicus, and Anaerococcus prowazekii.

[0040] Furthermore, the kit also includes dNTP(U)s, PCR buffer and Mg 2+ At least one of .

[0041] Furthermore, the kit also includes at least one of DNA polymerase and uracil glycosylase (UNG enzyme).

[0042] Furthermore, the kit also includes at least one of a nucleic acid releasing reagent and a nucleic acid extracting reagent.

[0043] Furthermore, the kit also includes a nucleic acid release reagent, a nucleic acid extraction reagent, dNTP, dUTP, uracil glycosylase (UNG enzyme), DNA polymerase, PCR buffer and Mg 2+ At least one of .

[0044] Furthermore, the concentration of the DNA polymerase is 5 U / reaction to 15 U / reaction, for example, the DNA polymerase may be Taq enzyme.

[0045] In a specific embodiment, the kit of the present invention comprises Taq enzyme, UNG enzyme, Mg 2+ , dNTP(U)s, primers, probes and PCR buffer.

[0046] The main components of PCR buffer include Tris-HCl, NaCl, NH4Cl, and Tween 20.

[0047] In some specific embodiments, the total volume of the reactants in a single PCR reaction tube is 20 μL to 60 μL.

[0048] 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.

[0049] In a fourth aspect, a method for detecting pathogens associated with wound infection is provided, the method comprising 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] In the present invention, the sample used for detection may be tissue fluid or wound exudate, etc., but is not limited thereto.

[0054] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0055] UNG decontamination, temperature is 50-65℃, time is 1-5 minutes, 1 cycle; pre-denaturation, temperature is 90-99℃, time is 0.5-6min, 1 cycle; denaturation, temperature is 90-99℃, time is 5-20 seconds, annealing, temperature is 55℃-60℃, time is 10-60 seconds, 30-50 cycles, and fluorescence collection.

[0056] Furthermore, the method for detecting pathogens associated with wound infection is for non-diagnostic purposes.

[0057] In a specific embodiment, a composition is provided for preparing a reagent for detecting pathogens associated with wound infection, wherein the detection comprises the following steps:

[0058] 1) Extracting nucleic acid from the sample to be tested;

[0059] 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);

[0060] 3) Obtain and analyze the results.

[0061] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0062] UNG decontamination, temperature is 50-65℃, time is 1-5 minutes, 1 cycle; pre-denaturation, temperature is 90-99℃, time is 0.5-6min, 1 cycle; denaturation, temperature is 90-99℃, time is 5-20 seconds, annealing, temperature is 55℃-60℃, time is 10-60 seconds, 30-50 cycles, and fluorescence collection.

[0063] In this article, the term "non-diagnostic purpose" means that it 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 above pathogens are present in the environment and culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figures 1 to 3 The results of testing different samples for the composition of the present invention are shown in FIG.

[0065] Figures 4 to 8 is a sensitivity result diagram of the composition of the present invention (respectively, Peptostreptococcus anaerobicus, Peptostreptococcus insaccharolyticus, and Anaerococcus prausnitzii);

[0066] Fig. 9 It is a graph showing the specific results of the composition of the present invention. DETAILED DESCRIPTION

[0067] 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.

[0068] Example 1: Primers and probes used in the present invention

[0069] The primers and probes used in the present invention are shown in Table 1 below:

[0070] Table 1

[0071]

[0072] Among them, the fluorescent reporter group of the probe for detecting Bacillus thuringiensis is FAM; the fluorescent reporter group of the probe for detecting Corynebacterium striata is HEX; the fluorescent reporter group of the probe for detecting anaerobic Streptococcus is ROX; the fluorescent reporter group of the probe for detecting insugar-insoluble Streptococcus is CY5; and the fluorescent reporter group of the probe for detecting Anaerobic Coccus provocateur is QUASAR 705.

[0073] Example 2: Method for detecting pathogens

[0074] Reagent preparation

[0075] 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 according to the proportion (PCR reaction solution 19 μL / person + enzyme mixture 1 μL / person), mix them thoroughly to form a PCR mixture, and centrifuge at 2000 rpm for 10 seconds before use.

[0076] The enzyme mixture consists of UNG enzyme and H-Taq enzyme. H-Taq enzyme (15U / μL) and UNG enzyme (5U / μL) are mixed in a certain ratio (0.9μL H-Taq enzyme and 0.1μL UNG enzyme per person).

[0077] The PCR reaction solution was prepared according to Table 2.

[0078] Table 2

[0079] Components The volume of each reaction PCR buffer* 15.80μL dNTPs(100mM) 0.50μL <![CDATA[1mol / L MgCl2]]> 0.20μL Primer (50pmol / μL) 0.2μL / strip Probe (50pmol / μL) 0.1μL / strip

[0080] Sample handling and loading

[0081] 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.

[0082] 5 μL of the above-treated samples, negative controls and positive controls were pipetted and added to the corresponding 0.2 mL PCR reaction tubes, 20 μL of PCR mixture was added to each tube, and the tubes were capped.

[0083] PCR amplification

[0084] PCR amplification was performed on a SLAN-96P fully automatic medical PCR analyzer according to the temperature and time setting program shown in Table 3.

[0085] Table 3

[0086]

[0087]

[0088] Result analysis and judgment

[0089] 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 Dafengoldensis; 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 Corynebacterium striata; 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 anaerobic Peptostreptococcus; 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 Peptostreptococcus saccharolyticus; 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 Anaerobic Coccus Provost; if the sample FAM, HEX, ROX, CY5 and QUASAR 705 channels have no amplification curve (No Ct) or the Ct value is greater than 40, it is judged to be negative. The details are shown in Table 4 below.

[0090] Table 4

[0091]

[0092] Example 3: Test results of the test samples of the composition of the present invention

[0093] The primers and probes shown in Example 1 were used to perform PCR detection on a mixed sample of Peptostreptococcus anaerobicus, Peptostreptococcus insaccharolyticus according to the method of Example 2 on a SLAN-96P real-time fluorescence quantitative PCR instrument. The results showed that the composition of the present invention can detect and distinguish any combination of the above-mentioned related pathogens. For example, the composition of the present invention detects:

[0094] Corynebacterium striata, anaerobic Peptostreptococcus, insaccharolytic Peptostreptococcus, Anaerobic Coccus prausnitzii ( Figure 1 );

[0095] Peptostreptococcus anaerobicus, Peptostreptococcus insaccharolyticus, Anaerobic coccus prausnitzii ( Figure 2 );

[0096] Finegold bacteria, Corynebacterium striata, Peptostreptococcus anaerobicus, Peptostreptococcus insaccharolyticus, Anaerobic coccus prausnitzii ( Figure 3 ).

[0097] These figures can prove that the present invention is a 5-plex PCR that can amplify the above-mentioned 5 targets at one time and can be distinguished using a 5-channel PCR instrument. It also proves that any corresponding pair combination of the composition of the present invention can detect any corresponding combination of pathogens.

[0098] Example 4: Sensitivity of the composition of the present invention

[0099] Using the composition in Example 1 of the present invention, the plasmids were diluted in gradients, and the concentrations were 500000, 50000, 5000, and 500 copies / mL, respectively, and the four concentrations were tested. Among them, the test results of the plasmid samples of 500 copies / mL of Bacillus fergifer, Corynebacterium striata, Peptostreptococcus anaerobicus, Peptostreptococcus insaccharolyticus, and Anaerococcus provocateuronas were as follows: Figure 4-8 The results show 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.

[0100] Example 5. Specificity of the composition of the present invention

[0101] The composition of the present invention has no cross reaction with pathogens related to wound infection (Fusobacterium nucleatum, Lactobacillus acidophilus, Propionibacterium freudenreichii, Bifidobacterium, Clostridium perfringens, Veillonella). Fig. 9 As shown in Table 5, it is shown that the composition of the present invention has good specificity.

[0102] Table 5

[0103]

[0104]

[0105] Example 6: Anti-interference and stability of the composition of the present invention

[0106] Potential PCR inhibitors / interfering substances such as oxymetazoline hydrochloride, dexamethasone, cefmenoxime hydrochloride, benzocaine, zanamivir, ribavirin, azithromycin, sodium chloride, SDS, EDTA, urea, heme, purified mucin, anhydrous ethanol, mupirocin, tobramycin, etc. at a certain concentration had no obvious effect on the composition of the present invention. The results are shown in Table 6.

[0107] Table 6

[0108]

[0109] 10 Stability studies have shown that the composition of the present invention can be stored for 11 months under actual storage conditions (-20±5°C).

[0110] After testing for 48 hours, the performance was stable; the results of the 37°C accelerated destruction stability experiment showed that the composition of the present invention was stored in a 37°C constant temperature box for 48 hours, and the results met the quality requirements; the freeze-thaw stability test showed that the composition of the present invention was frozen and thawed once at each test time point at the actual storage temperature, and was tested 5 times in a row, and the results all met the quality requirements.

Claims

1. A composition for detecting pathogens associated with wound infection, comprising upstream and downstream primers and probes for detecting at least 4 of the following pathogens: Upstream primers, downstream primers and probes for detecting Bacillus thuringiensis as shown in SEQ ID NOs: 1 to 3; Upstream primers, downstream primers and probes for detecting Corynebacterium striatum as shown in SEQ ID NOs: 4 to 6; Upstream primers, downstream primers and probes for detecting anaerobic peptostreptococci as shown in SEQ ID NOs: 7 to 9; The upstream primers, downstream primers and probe for detecting Peptostreptococcus insaccharolyticus as shown in SEQ ID NOs: 10 to 12; or The upstream primers, downstream primers and probe for detecting Anaerococcus provocateurus are shown in SEQ ID NOs: 13-15.

2. The composition according to claim 1, characterized in that The composition comprises: Upstream primers, downstream primers and probes for detecting Bacillus thuringiensis as shown in SEQ ID NOs: 1 to 3; Upstream primers, downstream primers and probes for detecting Corynebacterium striatum as shown in SEQ ID NOs: 4 to 6; Upstream primers, downstream primers and probes for detecting anaerobic peptostreptococci as shown in SEQ ID NOs: 7 to 9; Upstream primers, downstream primers and probes for detecting Peptostreptococcus insaccharolyticus as shown in SEQ ID NOs: 10 to 12; and The upstream primers, downstream primers and probe for detecting Anaerococcus provocateurus are shown in SEQ ID NOs: 13-15.

3. The composition according to claim 2, characterized in that The fluorescent groups between the probes detecting different pathogens are different and do not interfere with each other.

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 pathogens associated with wound infection, wherein: The pathogen is one or more of Peptostreptococcus insaccharolyticus, Peptostreptococcus ...anaerobicus, or Anaerococcus prausnitzii.

7. A kit for detecting pathogens associated with wound infection, the kit 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, a UNG enzyme, dNTP, dUTP, a PCR buffer, or Mg 2+ At least one of .

10. Use of a composition for preparing a reagent for detecting pathogens associated with wound infection, 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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