Compositions, kits and uses for detecting wound infection-associated pathogens

Abstract

The present application belongs to the field of molecular biology detection, and particularly relates to detection of wound infection related pathogens, more particularly to detection of at least four types of pathogens in Streptococcus, Bacteroides fragilis, Bacteroides, Conidiobolus corruptus and Red hair moss fungus, so as to determine the infection source as early as possible and provide corresponding prevention and control measures in a targeted and timely manner. The composition of the present application has higher detection sensitivity of 500 copies / mL, good specificity, more accurate detection, can provide sufficient rapid detection and basis for excluding infection of different pathogens, shortens the judgment time of wound infection causes, and accelerates the implementation of response measures.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology detection, specifically, it relates to the detection of pathogens related to wound infection, and more specifically, it relates to the detection of *Peptostreptococcus*, *Bacteroides fragilis*, *Bacteroides*, *Fusarium solani*, and *Trichophyton rubrum*. Background Technology

[0002] *Peptostreptococcus* spp. is a genus of Gram-positive bacteria belonging to the family Peptostreptococciaceae in the order Eubacteria. These bacteria are obligate parasites of the oral cavity, mucous membranes, and intestines of mammals and may play a role in purulent infections. *Peptostreptococcus* are commensal bacteria in humans, primarily residing in the oral cavity, skin, gastrointestinal tract, vagina, and urinary tract, and are members of the gut microbiota. Under immunosuppression or traumatic conditions, these bacteria can become pathogenic, leading to sepsis and harming the host. *Peptostreptococcus* can cause brain, liver, breast, and lung abscesses, as well as systemic necrotizing soft tissue infections. They are involved in mixed anaerobic infections, infections caused by a variety of bacteria that do not require or may be harmed by oxygen.

[0003] Bacteroides spp. is a genus of anaerobic, Gram-negative rod-shaped bacteria. It is part of the normal human colonic microbiota and is usually a symbiotic organism, but can cause infection if it enters the bloodstream or surrounding tissues after surgery, illness, or trauma. Bacteroides fragilis is a representative strain of the genus, and due to its adhesiveness, hemagglutinin, polysaccharide capsules, and fimbriae, it is the most pathogenic of the genus, causing intra-abdominal infections, postoperative wound infections, diabetic foot infections, and bacteremia.

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

[0005] Trichophyton rubrum is a dermatophyte belonging to the phylum Ascomycota. It is a clonal, saprophytic fungus that lives on the surface of dead skin and is the most common cause of athlete's foot, nail fungus, and tinea cruris worldwide.

[0006] Wound infections are not only common, but they can also recur, be chronic, and even fatal. Using the right tools for specialized research to determine the cause of infection is crucial for effectively combating wound infections. Therefore, for infected patients, it is essential to identify the source of infection as early as possible and provide targeted and timely treatment.

[0007] Therefore, there is a need in the field for a product that can easily and quickly detect the aforementioned pathogens, and has good sensitivity and specificity, capable of detecting and distinguishing the pathogenic microorganisms that cause infection, so as to provide targeted strategies for the prevention and control of the aforementioned wound infections. Summary of the Invention

[0008] In view of this, in a first aspect, the present invention provides a composition for detecting wound infection-related pathogens, comprising upstream and downstream primers and probes for detecting at least four of the following pathogens:

[0009] For example, the upstream primer, downstream primer and probe for detecting Peptostreptococcus spp. shown in SEQ ID NO:1-3;

[0010] The upstream primers, downstream primers, and probes for detecting Bacteroides fragilis are shown in SEQ ID NO:4–6.

[0011] For example, the upstream primer, downstream primer and probe for detecting Bacteroides shown in SEQ ID NO:7~9;

[0012] For example, the upstream primers, downstream primers, and probes for detecting Fusarium solani, as shown in SEQ ID NO:10-12; or

[0013] The upstream primer, downstream primer, and probe for detecting Trichophyton rubrum are shown in SEQ ID NO:13-15.

[0014] The combined detection composition provided by this invention mainly utilizes multiplex fluorescent PCR analysis to detect different pathogens by detecting target sites on different pathogens. This allows for the simultaneous detection and differentiation of at least four types of pathogens—including *Peptostreptococcus*, *Bacteroides fragilis*, *Bacteroides*, *Fusarium solani*, and *Trichophyton rubrum*—in a single-tube reaction system. This enables early identification of the source of infection and timely provision of targeted prevention and control measures. The composition of this invention offers higher detection sensitivity (up to 500 copies / mL), better specificity, and more accurate detection. It provides sufficient and rapid detection and exclusion of different pathogen infections, shortening the time required to determine the cause of wound infection and accelerating the implementation of countermeasures.

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

[0016] For example, the upstream primer, downstream primer and probe for detecting Peptostreptococcus spp. shown in SEQ ID NO:1-3;

[0017] The upstream primers, downstream primers, and probes for detecting Bacteroides fragilis are shown in SEQ ID NO:4–6.

[0018] For example, the upstream primer, downstream primer and probe for detecting Bacteroides shown in SEQ ID NO:7~9;

[0019] For example, the upstream primers, downstream primers, and probes for detecting Fusarium solani, as shown in SEQ ID NO:10–12; and

[0020] The upstream primer, downstream primer, and probe for detecting Trichophyton rubrum are shown in SEQ ID NO: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 implementation schemes, an exogenous internal standard is added to the sample; furthermore, the internal standard is a human internal standard gene.

[0023] In one specific implementation plan, 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, "dissimilar and non-interfering" means that each probe in the composition uses a different fluorescent group, and these groups do 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. These groups have different absorbance values, allowing for the selection of different channels and thus preventing mutual interference.

[0026] In some specific implementations, 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] Furthermore, in some embodiments, the composition of the present invention may simultaneously comprise one or more sets of the primer and probe pairs described above. In the present invention, a "set" refers to a mutually matched upstream primer, downstream primer, and probe for detecting a target.

[0028] The compositions of this invention can be arbitrarily combined to detect any combination of five corresponding targets. Those skilled in the art can combine them as needed, determining which targets to detect by combining the primer and probe pairs corresponding to those targets. All such combinations are included in this invention.

[0029] For example, it may include any 4 sets of the above 5 sets of primers and probes, any 3 sets of the above 5 sets of primers and probes, any 2 sets of the above 5 sets of primers and probes, or any 1 set 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 quenching group, such as BHQ1 or BHQ2.

[0033] In one specific implementation, the quenching group at the 3' end of the probe is BHQ1.

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

[0035] In one specific embodiment, the components of the composition of the present invention are contained in the same package.

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

[0037] Secondly, the present invention provides the use of the above-described composition of the present invention in the preparation of a kit for detecting pathogens associated with wound infection, wherein the pathogen is one or more of Peptostreptococcus, Bacteroides fragilis, Bacteroides spp., Fusarium solani, or Trichophyton rubrum.

[0038] Thirdly, the present invention provides a kit for detecting pathogens associated with wound infection, the kit comprising the composition of the present invention as described above.

[0039] Furthermore, the kit also includes negative and positive controls.

[0040] In one specific implementation, the negative control is at least one of DEPC H2O and physiological saline; the positive control is at least one of fragment plasmids or positive strains of Peptostreptococcus, Bacteroides fragilis, Bacteroides spp., Fusarium solani, and Trichophyton rubrum.

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

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

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

[0044] Furthermore, the kit also includes nucleic acid release reagents, nucleic acid extraction reagents, dNTPs, dUTPs, uracil glycosylase (UNG enzyme), DNA polymerase, PCR buffer, and Mg... 2+ At least one of them.

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

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

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

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

[0049] In one specific implementation, the kit of the present invention is compatible with digital PCR amplification systems, that is, it can be directly used for amplification on a digital PCR instrument.

[0050] Fourthly, a method for detecting wound infection-related pathogens is provided, the method comprising the following steps:

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

[0052] 2) Perform fluorescent PCR detection on the nucleic acid obtained in step 1) using the composition of the present invention as described above or the kit of the present invention as described above;

[0053] 3) Obtain and analyze the results.

[0054] In this invention, the sample used for detection can be urine, wound abscess, wound tissue fluid, etc., but is not limited to these.

[0055] Furthermore, the reaction conditions for the real-time PCR are as follows:

[0056] UNG decontamination: temperature 50–65℃, time 1–5 minutes, 1 cycle; pre-denaturation: temperature 90–99℃, time 0.5–6 minutes, 1 cycle; denaturation: temperature 90–99℃, time 5–20 seconds; annealing: temperature 55℃–60℃, time 10–60 seconds, 30–50 cycles; fluorescence collection.

[0057] Furthermore, the method for detecting wound infection-related pathogens is not for diagnostic purposes.

[0058] In one specific embodiment, the use of a composition for preparing a reagent for detecting wound infection-related pathogens is provided, the detection comprising the following steps:

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

[0060] 2) Perform fluorescent PCR detection on the nucleic acid obtained in step 1) using the composition or kit of the present invention as described above;

[0061] 3) Obtain and analyze the results.

[0062] Furthermore, the reaction conditions for the real-time PCR are as follows:

[0063] UNG decontamination: temperature 50–65℃, time 1–5 minutes, 1 cycle; pre-denaturation: temperature 90–99℃, time 0.5–6 minutes, 1 cycle; denaturation: temperature 90–99℃, time 5–20 seconds; annealing: temperature 55℃–60℃, time 10–60 seconds, 30–50 cycles; fluorescence collection.

[0064] In this article, the term "non-diagnostic purpose" refers to methods not intended to obtain information about whether an individual is infected with the aforementioned pathogens and is suffering from an infection. For example, the method can detect the presence of the aforementioned pathogens in the environment and in cultures. Attached Figure Description

[0065] Figures 1-3 The results of testing different samples with the composition of the present invention are shown in the figure;

[0066] Figures 4-8 The sensitivity results of the compositions of the present invention are shown in the graphs (for *Streptococcus*, *Bacteroides fragilis*, *Bacteroides*, *Fusarium solani*, and *Trichophyton rubrum*, respectively).

[0067] Figure 9 This is a graph showing the specific results of the composition of the present invention. Detailed Implementation

[0068] The present invention will be described in detail below with reference to specific implementation schemes and embodiments, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific implementation schemes and embodiments are for illustrative purposes only and are not intended to limit the present invention.

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

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

[0071] Table 1

[0072]

[0073]

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

[0075] Example 2: Method for detecting pathogens

[0076] Reagent preparation

[0077] Based on the number of samples to be tested, positive controls, and negative controls, take the corresponding amounts of PCR reaction solution and enzyme mixture according to the ratio (14.6 μL / person for PCR reaction solution + 1.4 μL / person for enzyme mixture), mix thoroughly to form a PCR mixture, centrifuge at 2000 rpm for 10 seconds, and set aside for later use.

[0078] Preparation of enzyme mixture:

[0079] The enzyme mixture consists of UNG enzyme and H-Taq enzyme. H-Taq enzyme (15 U / μL) and UNG enzyme (5 U / μL) are mixed in a certain ratio (each person's dose is 1.15 μL of H-Taq enzyme and 0.25 μL of UNG enzyme).

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

[0081] Table 2

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

[0083] Sample processing 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 perform nucleic acid extraction using the nucleic acid extraction or purification reagents of Sansure Biotech Inc. in accordance with their instructions.

[0085] Take 4 μL of the prepared sample, negative control and positive control and add them to the corresponding 0.2 mL PCR reaction tubes. Add 16 μL of PCR mixture to each tube and cap the tubes.

[0086] PCR amplification

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

[0088] Table 3

[0089]

[0090] Results Analysis and Judgment

[0091] If the sample's FAM channel shows a distinct S-shaped amplification curve and the Ct value is ≤40, it is considered positive for *Peptostreptococcus* (general). If the sample's HEX channel shows a distinct S-shaped amplification curve and the Ct value is ≤40, it is considered positive for *Bacteroides fragilis*. If the sample's ROX channel shows a distinct S-shaped amplification curve and the Ct value is ≤40, it is considered positive for *Bacteroides*. If the sample's CY5 channel shows a distinct S-shaped amplification curve and the Ct value is ≤40, it is considered positive for *Fusarium solani*. If the sample's CY7 channel shows a distinct S-shaped amplification curve and the Ct value is ≤40, it is considered positive for *Trichophyton rubrum*. If the sample's FAM, HEX, ROX, CY5, and CY7 channels show no amplification curve (No Ct) or the Ct value is >40, it is considered negative. See Table 4 below for details.

[0092] Table 4

[0093]

[0094] Example 3: Detection results of 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 *Streptococcus* spp., *Bacteroides fragilis*, *Bacteroides* spp., *Fusarium solani*, and *Trichophyton rubrum* on a SLAN-96P real-time quantitative PCR instrument, following 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 can detect:

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

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

[0098] Oral peptic streptococci (verified as Pepticus genus), Bacteroides fragilis, Bacteroides commonis (Bacteroides genus), Fusarium solani, Trichophyton rubrum ( Figure 3 ).

[0099] These figures demonstrate that the present invention is a five-fold PCR that amplifies the above five targets in a single operation and can be distinguished using a five-channel PCR instrument. They also demonstrate that any combination of the corresponding combinations of the present invention's compositions can detect any combination of corresponding pathogens.

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

[0101] Using the composition in Example 1 of this invention, serially diluted plasmids were taken at concentrations of 500,000, 50,000, 5,000, and 500 copies / mL, and the four concentrations were tested. The detection results for the 500 copies / mL plasmid samples of *Peptostreptococcus canis* (*Peptostreptococcus* spp.), *Bacteroides fragilis*, *Bacteroides multiforme* (*Bacteroides* spp.), *Fusarium solani*, and *Trichophyton rubrum* are as follows: Figure 4-8 As shown. The results indicate that samples with concentrations as low as 500 copies / mL can still be accurately detected in each channel with a detection rate of 100%, demonstrating 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 this invention showed no cross-reactivity with other pathogens similar to wound infection-related pathogens (Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus lactis, Streptococcus salivarius, Streptococcus pyogenes, Trichophyton violaceum). Results are as follows... Figure 9 As shown in Table 5, the compositions of the present invention have excellent specificity.

[0104] Table 5

[0105]

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

[0107] Analysis of interfering substances showed that certain concentrations of potential PCR inhibitors / interfering substances such as oxymetazoline hydrochloride, dexamethasone, cefotaxime hydrochloride, benzocaine, zanamivir, ribavirin, azithromycin, sodium chloride, SDS, EDTA, urea, heme, purified mucin, anhydrous ethanol, mupirocin, and tobramycin had no significant effect on this kit. The results are shown in Table 6.

[0108] Table 6

[0109]

[0110] Stability studies show that the composition of the present invention is stable after being stored under actual storage conditions (-20±5℃) for 11 months. The accelerated degradation stability test at 37℃ shows that the composition of the present invention meets the quality requirements after being stored in a constant temperature chamber at 37℃ for 24 hours. The freeze-thaw stability test shows that different batches of the composition of the present invention, after undergoing one freeze-thaw cycle at each test time point under actual storage temperature, and after four consecutive tests, all meet the quality requirements.

Claims

1. A composition for detecting wound infection-associated pathogens, characterized by, The composition comprises upstream primers, downstream primers and probes for detecting at least four of the following pathogens: upstream primers, downstream primers and probes for detecting Streptococcus as shown in SEQ ID NO: 1~3; upstream primers, downstream primers and probes for detecting Bacteroides fragilis as shown in SEQ ID NO: 4~6; upstream primers, downstream primers and probes for detecting Bacteroides as shown in SEQ ID NO: 7~9; upstream primers, downstream primers and probes for detecting Fusarium solani as shown in SEQ ID NO: 10~12; or upstream primers, downstream primers and probes for detecting Leptospaeria rubinnea as shown in SEQ ID NO: 13~15.

2. The composition of claim 1, wherein, The composition comprises: upstream primers, downstream primers and probes for detecting Streptococcus as shown in SEQ ID NO: 1~3; upstream primers, downstream primers and probes for detecting Bacteroides fragilis as shown in SEQ ID NO: 4~6; upstream primers, downstream primers and probes for detecting Bacteroides as shown in SEQ ID NO: 7~9; upstream primers, downstream primers and probes for detecting Fusarium solani as shown in SEQ ID NO: 10~12; and upstream primers, downstream primers and probes for detecting Leptospaeria rubinnea as shown in SEQ ID NO: 13~15.

3. The composition of claim 2, wherein, The fluorescent groups of the probes for detecting different pathogens are different from each other and do not interfere with each other.

4. The composition according to any one of claims 1 to 3, characterized in that, The composition further comprises upstream primers, downstream primers and probes for detecting an internal standard.

5. The composition of claim 4, wherein, The components of the composition are in a mixed form.

6. Use of a composition according to any one of claims 1 to 5 for the manufacture of a kit for the detection of a pathogen associated with a wound infection, characterized in that, The pathogens are one or more of Fusarium solani, Streptococcus, Bacteroides fragilis, Bacteroides, or Leptospaeria rubinnea.

7. A kit for detecting wound infection-associated pathogens, characterized in that, The kit comprises the composition of any one of claims 1~5.

8. The kit of claim 7, wherein The kit further comprises negative and positive quality controls. The kit further comprises negative and positive quality controls.

9. The kit according to claim 7 or 8, characterized in that, The kit further comprises at least one of: a nucleic acid release reagent, a nucleic acid extraction reagent, a DNA polymerase, a UNG enzyme, dNTPs, dUTP, a PCR buffer, or Mg 2+ ​

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