Composition for detecting wound infection related pathogens, kit and application

Abstract

The invention belongs to the field of molecular biological detection, particularly relates to detection of pathogens related to wound infection, and more particularly relates to detection of at least four pathogens in peptostreptococcus, bacteroides fragilis, bacteroides, fusarium solani and trichothecium rubrum, so that an infection source is determined as early as possible, and corresponding prevention and treatment measures are provided in time in a targeted manner. 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, Bacteroides fragilis, Bacteroides, Fusarium solani, and Trichophyton rubrum. Background Art

[0002] Peptostreptococcus spp. is a genus of Gram-positive bacteria in the family Peptostreptococci of the order Eubacteriales. Species of this genus are obligate parasites of the oral cavity, mucus membranes, and intestines of mammals and may play a role in suppurative infections. Peptostreptococci are commensal bacteria of humans, living primarily in the oral cavity, skin, gastrointestinal tract, vagina, and urinary tract as members of the intestinal flora. Under conditions of immunosuppression or trauma, these bacteria become pathogenic and cause sepsis, harming the host. Peptostreptococci can cause abscesses of the brain, liver, breast, and lung, as well as necrotizing soft tissue infections throughout the body. They are involved in mixed anaerobic infections, i.e. infections caused by a variety of bacteria that do not require oxygen and may not even be harmed by it.

[0003] Bacteroides spp. is an anaerobic, Gram-negative rod-shaped bacterium. It is part of the normal microbiota of the human colon and is usually a commensal, but can cause infection if it enters the blood or surrounding tissues after surgery, disease, or trauma. Bacteroides fragilis is a representative strain of the genus Bacteroides. Due to its adhesion, hemagglutinin, polysaccharide capsule, pili and other virulence factors, it has the highest pathogenicity in the genus Bacteroides and can cause abdominal infection, postoperative wound infection, diabetic foot infection, bacteremia, etc.

[0004] Fusarium solani is a saprophytic fungus that is associated with soft tissue infections, keratitis, and onychomycosis in immunocompetent hosts. Fusarium solani can cause infections in the skin, mucous membranes, respiratory tract, and other sites, and the infections can be localized or disseminated.

[0005] Trichophyton rubrum is a dermatophyte of the phylum Ascomycota. It is a monoclonal, anthropophilic saprophyte that lives in the upper layers of dead skin and is the most common cause of athlete's foot, fungal nail infections, and tinea cruris worldwide.

[0006] Wound infections are not only common, but can also be recurrent, chronic, and even fatal. Using the right tools to conduct specialized studies to determine the cause of infection is key to effectively combating wound infections. Therefore, for infected patients, the source of infection must be identified as early as possible, and targeted and timely countermeasures must be provided.

[0007] 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 pathogenic microorganisms that cause infection, so as to provide targeted strategies for the prevention and treatment of the above-mentioned wound infections. Summary of the invention

[0008] 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:

[0009] Upstream primers, downstream primers and probes for detecting Peptostreptococcus as shown in SEQ ID NOs: 1 to 3;

[0010] Upstream primers, downstream primers and probes for detecting Bacteroides fragilis as shown in SEQ ID NOs: 4 to 6;

[0011] Upstream primers, downstream primers and probes for detecting Bacteroides as shown in SEQ ID NOs: 7 to 9;

[0012] The upstream primers, downstream primers and probe for detecting Fusarium solani as shown in SEQ ID NOs: 10 to 12; or

[0013] The upstream primers, downstream primers and probe for detecting Trichophyton rubrum are shown in SEQ ID NOs: 13-15.

[0014] 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 types of pathogens in the genus Peptostreptococcus, Bacteroides fragilis, Bacteroides genus, Fusarium solani, and Trichophyton rubrum in a single-tube reaction system, so as to clarify the source of infection as early as possible, and provide corresponding prevention and treatment measures in a targeted and timely manner. 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.

[0015] Furthermore, the present invention provides a composition for detecting pathogens related to wound infection, comprising:

[0016] Upstream primers, downstream primers and probes for detecting Peptostreptococcus as shown in SEQ ID NOs: 1 to 3;

[0017] Upstream primers, downstream primers and probes for detecting Bacteroides fragilis as shown in SEQ ID NOs: 4 to 6;

[0018] Upstream primers, downstream primers and probes for detecting Bacteroides as shown in SEQ ID NOs: 7 to 9;

[0019] Upstream primers, downstream primers and probes for detecting Fusarium solani as shown in SEQ ID NOs: 10 to 12; and

[0020] The upstream primers, downstream primers and probe for detecting Trichophyton rubrum are shown in SEQ ID NOs: 13-15.

[0021] Furthermore, the composition includes an upstream primer, a downstream primer and a probe for detecting an internal standard.

[0022] In some specific embodiments, an exogenous internal standard is added to the sample, and further, the internal standard is a human internal standard gene.

[0023] In a specific embodiment, the internal standard is RNase P or GAPDH.

[0024] 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.

[0025] 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, CY7 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.

[0026] In some specific embodiments, the fluorescent reporter group of the probe for detecting Peptostreptococcus is FAM; the fluorescent reporter group of the probe for detecting Bacteroides fragilis is HEX; the fluorescent reporter group of the probe for detecting Bacteroides is ROX; the fluorescent reporter group of the probe for detecting Fusarium solani is CY5; and the fluorescent reporter group of the probe for detecting Trichophyton rubrum is CY7.

[0027] 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.

[0028] The composition of the present invention can be combined into any combination of detection corresponding to 5 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.

[0029] 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.

[0030] In some specific embodiments, the compositions of the present invention are used for fluorescent PCR.

[0031] Furthermore, the 3' end of the probe also has a non-fluorescent quencher.

[0032] Furthermore, the 3' end of the probe also has a quencher group, such as BHQ1 or BHQ2.

[0033] In a specific embodiment, the quencher group at the 3' end of the probe is BHQ1.

[0034] In a specific embodiment, each component of the composition of the present invention is present in a separate package.

[0035] In a particular embodiment, the components of the composition of the present invention are present in the same package.

[0036] Furthermore, the components of the composition of the present invention are present in a mixed form.

[0037] 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, Bacteroides fragilis, Bacteroides, Fusarium solani, or Trichophyton rubrum.

[0038] 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.

[0039] Furthermore, the kit also includes a negative quality control product and a positive quality control product.

[0040] 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, Bacteroides fragilis, Bacteroides, Fusarium solani, and Trichophyton rubrum.

[0041] Furthermore, the kit also includes dNTP(U)s, PCR buffer and Mg 2+ At least one of .

[0042] Furthermore, the kit also includes at least one of DNA polymerase and uracil glycosylase (UNG enzyme).

[0043] Furthermore, the kit also includes at least one of a nucleic acid releasing reagent and a nucleic acid extracting reagent.

[0044] 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 .

[0045] Furthermore, the concentration of the DNA polymerase is 5 U / reaction to 15 U / reaction, for example, the DNA polymerase may be Taq enzyme.

[0046] In a specific embodiment, the kit of the present invention comprises Taq enzyme, UNG enzyme, MgCl2, dNTP(U)s, primers, probe and PCR buffer.

[0047] The main components of PCR buffer include Tris-HCl, NaCl, NH4Cl, and Tween 20.

[0048] In some specific embodiments, the total volume of the reactants in a single PCR reaction tube is 20 μL to 60 μL.

[0049] 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.

[0050] In a fourth aspect, a method for detecting pathogens associated with wound infection is provided, the method comprising the following steps:

[0051] 1) Extracting nucleic acid from the sample to be tested;

[0052] 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);

[0053] 3) Obtain and analyze the results.

[0054] In the present invention, the sample used for detection may be urine, wound abscess, wound tissue fluid, etc., but is not limited thereto.

[0055] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0056] 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.

[0057] Furthermore, the method for detecting pathogens associated with wound infection is for non-diagnostic purposes.

[0058] 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:

[0059] 1) Extracting nucleic acid from the sample to be tested;

[0060] 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);

[0061] 3) Obtain and analyze the results.

[0062] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0063] 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.

[0064] 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

[0065] Figures 1 to 3 The results of testing different samples for the composition of the present invention are shown in FIG.

[0066] Figures 4 to 8 is a sensitivity result diagram of the composition of the present invention (respectively Peptostreptococcus, Bacteroides fragilis, Bacteroides, Fusarium solani, and Trichophyton rubrum);

[0067] Fig. 9 It is a graph showing the specific results of the composition of the present invention. DETAILED DESCRIPTION

[0068] 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.

[0069] Example 1: Primers and probes used in the present invention

[0070] The primers and probes used in the present invention are shown in Table 1 below:

[0071] Table 1

[0072]

[0073]

[0074] Among them, the fluorescent reporter group of the probe for detecting Streptococcus is FAM; the fluorescent reporter group of the probe for detecting Bacteroides fragilis is HEX; the fluorescent reporter group of the probe for detecting Bacteroides is ROX; the fluorescent reporter group of the probe for detecting Fusarium solani is CY5; and the fluorescent reporter group of the probe for detecting Trichophyton rubrum is CY7.

[0075] Example 2: Method for detecting pathogens

[0076] Reagent preparation

[0077] 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 14.6 μL / person + enzyme mixture 1.4 μL / person), mix them thoroughly to form a PCR mixture, and centrifuge at 2000 rpm for 10 seconds before use.

[0078] Preparation of enzyme mixture:

[0079] 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 (1.15μL H-Taq enzyme and 0.25μL UNG enzyme per person).

[0080] The PCR reaction solution was prepared according to Table 2 below.

[0081] Table 2

[0082] Components The volume of each reaction PCR buffer 11.35μL dNTPs(100mM) 0.5μL <![CDATA[1mol / L MgCl2]]> 0.25μL Primer (50pmol / μL) 0.2μL / strip Probe (50pmol / μL) 0.1μL / strip Total volume 14.6μL

[0083] Sample handling and loading

[0084] 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.

[0085] 4 μL of the above-treated samples, negative controls and positive controls were pipetted and added to the corresponding 0.2 mL PCR reaction tubes, 16 μL of PCR mixture was added to each tube, and the tubes were capped.

[0086] PCR amplification

[0087] PCR amplification was performed on the SLAN-96P fully automatic medical PCR analysis system according to the temperature and time setting program shown in Table 3.

[0088] Table 3

[0089]

[0090] Result analysis and judgment

[0091] If the sample FAM channel has an obvious S-type amplification curve and the Ct value is ≤40, it is judged as Peptostreptococcus (universal) positive; if the sample HEX channel has an obvious S-type amplification curve and the Ct value is ≤40, it is judged as Bacteroides fragilis positive; if the sample ROX channel has an obvious S-type amplification curve and the Ct value is ≤40, it is judged as Bacteroides positive; if the sample CY5 channel has an obvious S-type amplification curve and the Ct value is ≤40, it is judged as Fusarium solani positive; if the sample CY7 channel has an obvious S-type amplification curve and the Ct value is ≤40, it is judged as Trichophyton rubrum positive; if the sample FAM, HEX, ROX, CY5 and CY7 channels have no amplification curve (No Ct) or the Ct value is greater than 40, it is judged as negative. The details are shown in Table 4 below.

[0092] Table 4

[0093]

[0094] Example 3: Test results of the test samples of the composition of the present invention

[0095] The primers and probes shown in Example 1 were used to perform PCR detection on a mixed sample of Peptostreptococcus, Bacteroides fragilis, Bacteroides, Fusarium solani, and Trichophyton rubrum on a SLAN-96P real-time fluorescence quantitative PCR instrument according to the method of Example 2. 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:

[0096] Bacteroides fragilis, Fusarium solani, Trichophyton rubrum ( Figure 1 );

[0097] Anaerobic Peptostreptococcus (verified Peptostreptococcus), Bacteroides vulgaris (verified Bacteroides), Fusarium solani, Trichophyton rubrum ( Figure 2 );

[0098] Oral Peptostreptococcus (Peptostreptococcus), Bacteroides fragilis, Bacteroides vulgaris (Bacteroides), Fusarium solani, Trichophyton rubrum ( Figure 3 ).

[0099] 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.

[0100] Example 4: Sensitivity of the composition of the present invention

[0101] Using the composition in Example 1 of the present invention, the gradient dilution plasmid was taken, and the concentrations were 500000, 50000, 5000, and 500 copies / mL, respectively, and the four concentrations were tested. Among them, the test results of 500 copies / mL canine peptostreptococcus (Peptostreptococcus), Bacteroides fragilis, Bacteroides thetaiotaomicron (Bacteroides), Fusarium solani, and Trichophyton rubrum plasmid samples 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.

[0102] Example 5. Specificity of the composition of the present invention

[0103] The composition of the present invention has no cross-reaction with other pathogens similar to wound infection-related pathogens (Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus uberis, Streptococcus salivarius, Streptococcus pyogenes, Trichophyton violaceum). Fig. 9 As shown in Table 5, it is shown that the composition of the present invention has good specificity.

[0104] Table 5

[0105]

[0106] Example 6: Anti-interference and stability of the composition of the present invention

[0107] The results of the interference analysis experiment showed that certain concentrations of 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. had no obvious effect on this kit. The results are shown in Table 6.

[0108] Table 6

[0109]

[0110] The stability study showed that the performance of the composition of the present invention was stable after being stored for 11 months under actual storage conditions (-20±5°C); 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 24 hours, and the results met the quality requirements; the freeze-thaw stability test showed that different batches of the composition of the present invention were frozen and thawed once at each test time point at the actual storage temperature, and were tested four times in a row, and the results all met the quality requirements.

Claims

1. A composition for detecting pathogens related to wound infection, characterized in that: The composition includes upstream and downstream primers and probes for detecting at least 4 of the following pathogens: Upstream primers, downstream primers and probes for detecting Peptostreptococcus as shown in SEQ ID NOs: 1 to 3; Upstream primers, downstream primers and probes for detecting Bacteroides fragilis as shown in SEQ ID NOs: 4 to 6; Upstream primers, downstream primers and probes for detecting Bacteroides as shown in SEQ ID NOs: 7 to 9; The upstream primers, downstream primers and probe for detecting Fusarium solani as shown in SEQ ID NOs: 10 to 12; or The upstream primers, downstream primers and probe for detecting Trichophyton rubrum 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 Peptostreptococcus as shown in SEQ ID NOs: 1 to 3; Upstream primers, downstream primers and probes for detecting Bacteroides fragilis as shown in SEQ ID NOs: 4 to 6; Upstream primers, downstream primers and probes for detecting Bacteroides as shown in SEQ ID NOs: 7 to 9; Upstream primers, downstream primers and probes for detecting Fusarium solani as shown in SEQ ID NOs: 10 to 12; and The upstream primers, downstream primers and probe for detecting Trichophyton rubrum 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, characterized in that: The pathogen is one or more of Fusarium solani, Peptostreptococcus, Bacteroides fragilis, Bacteroides spp., or Trichophyton rubrum.

7. A kit for detecting pathogens related to wound infection, characterized in that: The kit comprises 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, characterized in that: The detection comprises 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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