Primer probe composition, kit and detection method for simultaneously detecting skin soft tissue infection related pathogens and drug-resistant genes thereof

Abstract

The invention belongs to the technical field of molecular biology, and discloses a primer probe composition, a kit and a detection method for simultaneously detecting skin soft tissue infection related pathogens and drug-resistant genes thereof, and the primer probe composition for simultaneously detecting the skin soft tissue infection related pathogens and the drug-resistant genes thereof. The kit comprises a primer probe composition aiming at escherichia coli, klebsiella pneumoniae, staphylococcus aureus, streptococcus agalactiae, beta hemolytic streptococcus, burkholderia, clostridium perfringens, nocardia, a beta-lactamase drug-resistant gene, a methicillin drug-resistant gene, a carbapenemase drug-resistant gene and a vancomycin drug-resistant gene. The sequences are respectively shown as SEQ ID NO.1-45. The primer probe composition and the kit are high in specificity and high in detection sensitivity, various pathogens and various drug-resistant genes related to skin soft tissue infection can be simultaneously detected in one tube, and an efficient and convenient method is provided for clinical medication guidance of skin soft tissue infection diseases.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology, and in particular to a primer-probe combination, a kit and a detection method for simultaneously detecting pathogens related to skin and soft tissue infection and drug-resistant genes thereof. Background Art

[0002] Skin and soft tissue infections (SSTIs) are inflammatory diseases caused by purulent pathogens invading the epidermis, dermis, and subcutaneous tissue, and include a large class of necrotic infections involving the skin, subcutaneous fat, fascia, and muscle layers. The severity of such diseases varies greatly, ranging from mild superficial localized infections to life-threatening deep necrotizing soft tissue infections. The treatment and prognosis vary depending on the classification and grading of SSTIs.

[0003] At present, there is no unified classification standard for SSTI. In clinical practice, it is mainly divided into three categories according to the patient's etiology, site of onset and severity of the disease: superficial bacterial skin infection, secondary SSTI and necrotizing soft tissue infection. Among them, superficial bacterial skin infection: includes folliculitis, furuncle, carbuncle, impetigo, cellulitis, erysipelas and suppurative hidradenitis. Secondary SSTI: includes surgical incision infection, trauma infection, burn infection, bite infection, infected cysts and abscesses, diabetic foot ulcers and pressure sore infection. Necrotizing soft tissue infection: refers to lesions involving the skin and deep structures, characterized by necrosis of the skin, subcutaneous tissue, fascia or skeletal muscle. This type is relatively rare and difficult to diagnose, but the disease progresses rapidly and the mortality rate is very high, up to 30%.

[0004] SSTI itself is not contagious, but some bacteria, viruses and fungi can be transmitted through direct contact, indirect contact, respiratory tract, sexual contact, mother-to-child vertical transmission, etc. SSTI can be complicated by bacteremia, sepsis, septic shock, lymphadenitis, pneumonia, acute renal failure, multiple organ dysfunction syndrome (MODS), etc.

[0005] When patients experience symptoms such as redness, swelling, increased skin temperature, and pain in the skin and soft tissues, they should seek medical attention promptly. The doctor will perform a physical examination on the patient, as well as blood routine, blood biochemistry, pathogenic examination, urine routine, erythrocyte sedimentation rate examination, serological examination, pathological biopsy and other examinations. Pathogenic examination is the gold standard for bacterial diagnosis, including the culture, isolation and drug sensitivity testing of pathogenic bacteria. For superficial bacterial skin infections, routine examinations are not required, but for SSTI patients with prolonged course, recurrent attacks or ineffective antibacterial drug treatment, pathogenic examinations should be performed to strive to identify the pathogens early and guide further treatment based on the results of drug sensitivity tests.

[0006] Mild SSTI patients are treated with topical antibiotics, which are mainly used for superficial bacterial skin infections such as folliculitis, impetigo and recurrent furunculosis. Topical antibiotics act directly on the target skin site, stay in the local area for a long time, and have a good therapeutic effect on superficial epidermal or dermal infections, but the disadvantage is that they are prone to antibiotic resistance. Commonly used topical antibiotics include mupirocin ointment, bacitracin ointment, retapamulin ointment, etc. Generally speaking, mild SSTI patients can take oral medication, while severe infections require intravenous administration. Doctors will comprehensively consider the patient's general condition, infection site, comorbidities, clinical manifestations and grading, and select antimicrobial drugs for systemic treatment in a targeted manner, and select sensitive antibiotics for treatment based on the results of drug sensitivity tests. Commonly used drugs include penicillin, clindamycin, cefazolin, cephalexin, piperacillin-tazobactam, cefoperazone-sulbactam, meropenem, imipenem, gentamicin, tobramycin, vancomycin, etc.

[0007] At present, due to the abuse of antibiotics, bacterial resistance has increased. Infection with drug-resistant bacteria is the main reason for treatment failure and increased mortality in SSTI patients, which seriously threatens human life and health. Therefore, in routine diagnosis, it is more important to diagnose the type of pathogenic pathogens of skin and soft tissue infections while also diagnosing their drug resistance, so that doctors can give more reasonable and effective treatment plans.

[0008] Therefore, the prior art needs to be further improved. Summary of the invention

[0009] In response to the above problems, the present invention provides a primer-probe combination, a kit and a detection method for simultaneously detecting pathogens related to skin and soft tissue infections and their drug-resistant genes. The primer-probe combination and the kit can not only detect 8 pathogens related to skin and soft tissue infections, but also simultaneously detect the drug-resistant genes of these pathogens. The detection efficiency is high and the specificity is strong, which greatly improves the work efficiency.

[0010] To solve the above problems, this application provides the following technical solutions:

[0011] In the first aspect, the present application also provides a primer-probe combination for simultaneously detecting pathogens related to skin and soft tissue infections and their drug resistance genes, which includes primer-probe combinations for Escherichia coli ybbJ gene, Klebsiella pneumoniae mgrB gene, Staphylococcus aureus MsrB gene, Streptococcus agalactiae PG-mutase gene, beta-hemolytic Streptococcus IS3 gene, Burkholderia 16S rRNA gene, Clostridium perfringens SpaA gene, Nocardia secA1 gene, and extended-spectrum β-lactamase resistance genes, methicillin resistance genes, carbapenemase resistance genes, and vancomycin resistance genes.

[0012] The primer probe for detecting the ybbJ gene of Escherichia coli includes: an upstream primer ybbJ-F having a sequence as shown in SEQ ID NO.1, a downstream primer ybbJ-R having a sequence as shown in SEQ ID NO.2, and a fluorescent probe ybbJ-P having a sequence as shown in SEQ ID NO.3;

[0013] The primer probe for detecting mgrB gene of Klebsiella pneumoniae includes: an upstream primer mgrB-F having a sequence as shown in SEQ ID NO.4, a downstream primer mgrB-R having a sequence as shown in SEQ ID NO.5, and a fluorescent probe mgrB-P having a sequence as shown in SEQ ID NO.6;

[0014] The primer probe for detecting the MsrB gene of Staphylococcus aureus includes: an upstream primer MsrB-F whose sequence is shown in SEQ ID NO.7, a downstream primer MsrB-R whose sequence is shown in SEQ ID NO.8, and a fluorescent probe MsrB-P whose sequence is shown in SEQ ID NO.9;

[0015] The primer probe for detecting the PG-mutase gene of Streptococcus agalactiae comprises: an upstream primer PG-mutase-F having a sequence as shown in SEQ ID NO.10, a downstream primer PG-mutase-R having a sequence as shown in SEQ ID NO.11, and a fluorescent probe PG-mutase-P having a sequence as shown in SEQ ID NO.12;

[0016] The primer probe for detecting IS3 gene of beta-hemolytic streptococcus includes: an upstream primer IS3-F whose sequence is shown in SEQ ID NO.13, a downstream primer IS3-R whose sequence is shown in SEQ ID NO.14, and a fluorescent probe IS3-P whose sequence is shown in SEQ ID NO.15;

[0017] The primer probe for detecting the 16S rRNA gene of Burkholderia includes: an upstream primer 16S rRNA-F having a sequence as shown in SEQ ID NO.16, a downstream primer 16S rRNA-F having a sequence as shown in SEQ ID NO.17, and a fluorescent probe 16S rRNA-P having a sequence as shown in SEQ ID NO.18;

[0018] The primer probe for detecting the SpaA gene of Clostridium perfringens includes: an upstream primer SpaA-F having a sequence as shown in SEQ ID NO.19, a downstream primer SpaA-R having a sequence as shown in SEQ ID NO.20, and a fluorescent probe SpaA-P having a sequence as shown in SEQ ID NO.21;

[0019] The primer probe for detecting the Nocardia secA1 gene includes: an upstream primer secA1-F having a sequence as shown in SEQ ID NO.22, a downstream primer secA1-R having a sequence as shown in SEQ ID NO.23, and a fluorescent probe secA1-P having a sequence as shown in SEQ ID NO.24;

[0020] The primer probe for detecting the β-lactamase resistance gene TEM includes: an upstream primer TEM-F whose sequence is shown in SEQ ID NO.25, a downstream primer TEM-R whose sequence is shown in SEQ ID NO.26, and a fluorescent probe TEM-P whose sequence is shown in SEQ ID NO.27;

[0021] The primer probe for detecting the β-lactamase resistance gene SHV includes: an upstream primer SHV-F whose sequence is shown in SEQ ID NO.28, a downstream primer SHV-R whose sequence is shown in SEQ ID NO.29, and a fluorescent probe SHV-P whose sequence is shown in SEQ ID NO.30;

[0022] The primer probe for detecting the β-lactamase resistance gene CTX-M-9 includes: an upstream primer CTX-M-9-F having a sequence as shown in SEQ ID NO.31, a downstream primer CTX-M-9-R having a sequence as shown in SEQ ID NO.32, and a fluorescent probe CTX-M-9-P having a sequence as shown in SEQ ID NO.33;

[0023] The primer probe for detecting the methicillin resistance gene mecA comprises: an upstream primer mecA-F having a sequence as shown in SEQ ID NO.34, a downstream primer mecA-R having a sequence as shown in SEQ ID NO.35, and a fluorescent probe mecA-P having a sequence as shown in SEQ ID NO.36;

[0024] The primer probe for detecting the carbapenemase resistance gene KPC-2 comprises: an upstream primer KPC-2-F having a sequence as shown in SEQ ID NO.37, a downstream primer KPC-2-R having a sequence as shown in SEQ ID NO.38, and a fluorescent probe KPC-2-P having a sequence as shown in SEQ ID NO.38;

[0025] The primer probe for detecting the vancomycin resistance gene VanA includes: an upstream primer VanA-F whose sequence is shown in SEQ ID NO.40, a downstream primer VanA-R whose sequence is shown in SEQ ID NO.41, and a fluorescent probe VanA-P whose sequence is shown in SEQ ID NO.42;

[0026] The primer probe for detecting the vancomycin resistance gene VanB includes: an upstream primer VanB-F whose sequence is shown in SEQ ID NO.43, a downstream primer VanB-R whose sequence is shown in SEQ ID NO.44, and a fluorescent probe VanB-P whose sequence is shown in SEQ ID NO.45;

[0027] The 5' end of the fluorescent probe is labeled with a fluorescent reporter group, and the 3' end of the fluorescent probe is labeled with a fluorescent quencher group.

[0028] Optionally, the primer probe composition also includes a primer probe for detecting the internal reference RNaseP gene: an upstream primer RNaseP-F1 with a sequence as shown in SEQ ID NO.46, a downstream primer RNaseP-R1 with a sequence as shown in SEQ ID NO.47, and a fluorescent probe RNaseP-P1 with a sequence as shown in SEQ ID NO.48.

[0029] Optionally, in the primer-probe composition, the fluorescent reporter group is selected from FAM, ROX, CY5, and VIC; the fluorescent quencher group is selected from BHQ1, BHQ2, and BHQ3.

[0030] Optionally, the fluorescent reporter group of the pathogen gene is selected from FAM, ROX and VIC, and its fluorescent quenching group is selected from BHQ1 and BHQ2; the fluorescent reporter group of multiple antibiotic resistance genes uses CY5, and its fluorescent quenching group uses BHQ3.

[0031] Preferably, the 5' end of the fluorescent probes ybbJ-P, mgrB-P and RNasP-P is labeled with a reporter group FAM, and the 3' end is labeled with a quencher group BHQ1; the 5' end of the fluorescent probes MsrB-P, PG-mutase-P and IS3-P is labeled with a reporter group VIC, and the 3' end is labeled with a quencher group BHQ1; the 5' end of the fluorescent probes 16S rRNA-P, SpaA-P, secA1-P is labeled with a reporter group ROX, and the 3' end is labeled with a quencher group BHQ2; the 5' end of the fluorescent probes TEM-P, SHV-P, CTX-M-9-P, mecA-P, KPC-2-P, VanA-P and VanB-P is labeled with a reporter group CY5, and the 3' end is labeled with a quencher group BHQ3.

[0032] In a second aspect, the present application also provides a kit for simultaneously detecting pathogens related to skin and soft tissue infections and their drug-resistant genes, characterized in that it comprises the aforementioned primer-probe combination.

[0033] Optionally, the kit further comprises: DNA polymerase and PCR reaction buffer.

[0034] Optionally, the kit further comprises: a positive control substance and a negative control substance; the positive control substance comprises a standard recombinant plasmid carrying each gene; and the negative control substance is TE Buffer.

[0035] Optionally, the kit further comprises: a sample releasing agent.

[0036] The sample releaser comprises the following components: 1-5% by volume of Tween 20, 3-7% by volume of Triton X-100, 1-5 mM of guanidine isothiocyanate, 5-15 mM of tris(hydroxymethyl)aminomethane salt and 0.1-5 mM of disodium ethylenediaminetetraacetate, and the remainder is sterile water; the pH of the sample releaser is 7.4±0.2.

[0037] Preferably, the sample releaser comprises the following components: 2% by volume of Tween 20, 5% by volume of Triton X-100, 2mM of guanidine isothiocyanate, 10mM of tris(hydroxymethyl)aminomethane salt and 1mM of disodium ethylenediaminetetraacetate, with the remainder being sterile water; the pH of the sample releaser is 7.4±0.2.

[0038] In a third aspect, the present application also provides the use of the above-mentioned primer-probe combination in the preparation of a kit for detecting pathogens related to skin and soft tissue infections and their drug resistance genes.

[0039] In a fourth aspect, the present application also provides a method for simultaneously detecting pathogens related to skin and soft tissue infection and their drug resistance genes, which comprises the following steps:

[0040] S1, pre-treating the collected sample to be tested and adding it to a PCR reaction tube, wherein the PCR reaction tube also contains the primer-probe combination as claimed in claim 1 and a PCR reaction solution, and performing qPCR amplification after mixing; after the reaction is completed, collecting the fluorescence signal of each channel to obtain the Tm value of the melting curve of each channel;

[0041] S2. Determine whether the sample to be tested contains each pathogen based on the Tm value of the melting curve of each channel in the PCR reaction system.

[0042] Optionally, the specific analysis and judgment method of step S2 is:

[0043] When the Tm value of the FAM channel is 65 ± 1, it means that the biological sample contains E. coli;

[0044] When the Tm value of the FAM channel is 70 ± 1, it means that the biological sample contains Klebsiella pneumoniae;

[0045] When the Tm value of the VIC channel is 62 ± 1, it means that the biological sample contains Staphylococcus aureus;

[0046] When the Tm value of the VIC channel is 68 ± 1, it means that the biological sample contains Streptococcus agalactiae;

[0047] When the Tm value of the VIC channel is 72±1, it means that the biological sample contains beta-hemolytic streptococci;

[0048] When the ROX channel Tm value is 64 ± 1, it means that the biological sample contains Burkholderia;

[0049] When the ROX channel Tm value is 68 ± 1, it means that the biological sample contains Clostridium perfringens;

[0050] When the ROX channel Tm value is 70 ± 1, it means that the biological sample contains Nocardia;

[0051] When the Tm value of the CY5 channel is 70±1, it means that the biological sample is resistant to extended-spectrum β-lactamases;

[0052] When the Tm value of the CY5 channel is 63 ± 1, it means that the biological sample is resistant to methicillin;

[0053] When the Tm value of the CY5 channel is 59 ± 1, it means that the biological sample is resistant to carbapenemase;

[0054] When the Tm value of the CY5 channel is 66±1, it means that the biological sample is resistant to vancomycin.

[0055] On the basis of the above, it is further preferred that the specific analysis and judgment method of step S2 further includes:

[0056] When the Tm value of the FAM channel is 72±1, it means that human genes are detected in the biological sample and the sample sampling is qualified.

[0057] The above method of the present invention is based on the qPCR melting curve method, and the different Tm values ​​of the melting curves of each channel are used for different pathogen types and drug resistance gene types, so that the corresponding pathogen types and drug resistance genes can be judged by the specific numbers of the Tm values. This method is simple and easy to operate, with high efficiency, and can simultaneously realize the simultaneous detection of 8 skin and soft tissue infection-related pathogens and 7 different drug resistance genes, greatly simplifying the operation while reducing the amount of samples and reagents used.

[0058] Optionally, the above method further comprises the following steps:

[0059] S3. Based on the standard curve of the standard recombinant plasmid of each target gene, the content of the target pathogen in the sample to be tested can be obtained according to the fluorescence amplification Ct value of the sample to be tested.

[0060] The above method can not only qualitatively determine the type and drug-resistant genes of pathogens related to skin and soft tissue infection in the sample, but also can simultaneously obtain quantitative detection of the concentration of the infected pathogen by measuring the fluorescence amplification Ct value of the sample to be tested.

[0061] Optionally, in step S1, the pretreatment method of the sample to be tested is: use a swab to wipe the infected skin and soft tissue of a patient with skin and soft tissue infection, put the sampled swab into a sample release agent, vortex mix, and let stand at room temperature. The supernatant can be used as an amplification template for a PCR experiment.

[0062] Optionally, the mixing time is 20 to 50 seconds and the mixture is allowed to stand at room temperature for 3 to 10 minutes.

[0063] The sample release agent can be an existing sample release agent, which is used to quickly process the sample to be tested and release the nucleic acid in the sample for subsequent fluorescent PCR amplification. The optional scheme of the sample release agent is as mentioned above.

[0064] The present invention has the following beneficial effects:

[0065] 1. The present invention provides a primer-probe composition, a kit and a detection method for simultaneously detecting pathogens related to skin and soft tissue infections and their drug-resistant genes. The primer-probe composition and the kit can simultaneously detect pathogens related to skin and soft tissue infections, such as Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus agalactiae, beta-hemolytic Streptococcus, Burkholderia, Clostridium perfringens and Nocardia in one tube, overcoming the defects of low sensitivity and long time of pathogen culture in existing clinical detection methods.

[0066] 2. The qPCR melting curve method of the present invention has high detection sensitivity, and can stably detect 500 or 1000 copies / ml of pathogen nucleic acid at the lowest level. The method can specifically detect the types of Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus agalactiae, beta-hemolytic Streptococcus, Burkholderia, Clostridium perfringens, and Nocardia, and quantitatively determine their contents in samples. It has no cross-reaction with other samples such as Salmonella, Shigella, Acinetobacter baumannii, Haemophilus, Pseudomonas aeruginosa, Proteus, Streptococcus pneumoniae, and Staphylococcus epidermidis, and has good specificity.

[0067] 3. The above-mentioned kit and detection method of the present application not only detect the type of pathogen, but also realize the detection of seven drug-resistant genes (TEM, SHV, CTX-M-9, mecA, KPC-2, VanA, VanB) of four common drug-resistant type genes (extended-spectrum β-lactamase, methicillin, carbapenemase, vancomycin) with a single fluorescence channel. The gene detection covers a wide range, and the operator can judge the drug-resistant type according to the Tm value, providing an efficient and convenient method for clinical guidance of medication.

[0068] 4. The above method of the present invention provides a sample processing method for skin and soft tissue infections. It does not require blood sampling for testing, and does not require complicated sample nucleic acid extraction and purification. It only requires the sample release agent to treat the sampling swab for a few minutes, and the supernatant can be used as a template for subsequent experiments, which greatly shortens the sample processing time.

[0069] 5. The detection method described in the present invention has strong specificity, high sensitivity, high efficiency and safety. It has good detection effect on trace amounts of Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus agalactiae, beta-hemolytic Streptococcus, Burkholderia, Clostridium perfringens, Nocardia genome and extended-spectrum β-lactamase, methicillin, carbapenemase, and vancomycin resistance genes contained in samples. It can be used for clinical detection and molecular epidemiological investigation of the above-mentioned pathogens and resistance genes, and lays a foundation for the development of PCR commercial diagnostic kits for pathogens related to skin and soft tissue infections such as Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus agalactiae, beta-hemolytic Streptococcus, Burkholderia, Clostridium perfringens, and Nocardia and the above-mentioned multiple resistance genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 Melting curve results of Escherichia coli, Klebsiella pneumoniae and internal reference genes;

[0071] Figure 2 Melting curve results for Staphylococcus aureus, Streptococcus agalactiae, and beta-hemolytic Streptococcus;

[0072] Figure 3 Melting curve results for Burkholderia, Clostridium perfringens, and Nocardia;

[0073] Figure 4 Melting curve results of drug resistance genes TEM, SHV, CTX-M-9, mecA, KPC-2, VanA, and VanB;

[0074] Figure 5 Fluorescence PCR test results and regression curves of Escherichia coli and Klebsiella pneumoniae;

[0075] Figure 6Fluorescence PCR test results and regression curves of Staphylococcus aureus and Streptococcus agalactiae;

[0076] Figure 7 Fluorescence PCR test results and regression curves of beta-hemolytic Streptococcus and Burkholderia;

[0077] Figure 8 Fluorescence PCR test results and regression curves of Clostridium perfringens and Nocardia spp.

[0078] Fig. 9 Fluorescence PCR test results and regression curves of drug resistance genes TEM and SHV;

[0079] Fig.10 Fluorescence PCR detection results and regression curves of drug resistance genes CTX-M-9 and mecA;

[0080] Fig.11 Fluorescence PCR detection results and regression curves of drug resistance genes KPC-2 and VanA;

[0081] Fig.12 Fluorescence PCR detection results and regression curve of drug resistance gene VanB;

[0082] Fig.13 Kit-specific test results;

[0083] Fig.14 Comparative Example 1: Melting curve results of the kit for detecting Staphylococcus aureus, Streptococcus agalactiae, and beta-hemolytic Streptococcus;

[0084] Fig.15 Comparative Example 2: Melting curve results of the kit for detecting Burkholderia, Clostridium perfringens and Nocardia;

[0085] Fig.16 Comparative Example 3: Melting curve results of Escherichia coli, Klebsiella pneumoniae and human internal reference detected by the kit. DETAILED DESCRIPTION

[0086] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are conventional methods in the art unless otherwise specified.

[0087] Example 1 Preparation of the kit

[0088] This embodiment provides a primer-probe combination and a kit for simultaneously detecting eight skin and soft tissue infection-related pathogens and multiple drug-resistant genes.

[0089] 1. Design and synthesis of primer-probe combinations

[0090] In order to achieve the simultaneous detection of eight pathogens related to skin and soft tissue infections and their drug resistance genes, based on the existing publicly available human genes, gene conservative sequences of Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus agalactiae, beta-hemolytic Streptococcus, Burkholderia, Clostridium perfringens, and Nocardia, as well as extended-spectrum β-lactamase, methicillin, carbapenemase, and vancomycin resistance genes, fluorescent PCR melting curve technology was used as the detection method to select and target the target genes of pathogens (Escherichia coli ybbJ gene, Klebsiella pneumoniae mgrB gene, Staphylococcus aureus MsrB gene, Streptococcus agalactiae PG-mutase gene, beta-hemolytic Streptococcus IS3 gene, Burkholderia 16S Specific primer probes were designed based on the sequence characteristics of seven drug-resistant genes, including rRNA gene, Clostridium perfringens SpaA gene, Nocardia secA1 gene), extended-spectrum β-lactamase (ESBL) resistance genes (TEM, SHV, CTX-M-9), methicillin (MR) resistance gene (mecA), carbapenemase (KPC) resistance gene (KPC-2) and vancomycin (Van) resistance gene (VanA, VanB), and verified by NCBI Blast.

[0091] At the same time, a specific primer probe of the internal reference RNasP gene is designed. The internal reference control can show the occurrence of false negatives and is used to monitor the collection, transportation, extraction and amplification process of the sample to be tested to avoid the interference of false negatives on the test results.

[0092] Table 1 Primers and fluorescent probes

[0093]

[0094] 2. Establishment of Fluorescence Quantitative PCR for Detecting Pathogens Related to Skin and Soft Tissue Infections and Their Resistance Genes

[0095] 1. Experimental methods

[0096] (1) Preparation of the kit

[0097] The kit includes: the primer-probe combination mentioned above, a buffer (using 5x Hotstart HiTaq Buffer (Mg 2+Plus)), reaction solution (Hotstart HiTaq Master PCR Mix (25x)), positive control substance and negative control substance. Other embodiments may use other buffers and reaction solutions, which are not limited to the specific types of this embodiment.

[0098] The reaction solution includes the 15 sets of primer-probe compositions, an internal reference primer-probe composition, a PCR reaction buffer and RNase free H2O.

[0099] The positive control is: standard recombinant plasmid (see below for details), and the negative control is TE Buffer.

[0100] The kit also includes a sample release agent, which includes the following components: 2% by volume of Tween 20, 5% by volume of Triton X-100, 2mM of guanidine isothiocyanate, 10mM of tris(hydroxymethyl)aminomethane salt and 1mM of disodium ethylenediaminetetraacetate, and the remainder is sterile water; the pH of the sample release agent is 7.4±0.2.

[0101] (2) Establishment of a fluorescence quantitative PCR method for detecting pathogens related to skin and soft tissue infections and their drug resistance genes using this kit

[0102] S1. Preparation of standard recombinant plasmid

[0103] The above pair of primer probes was designed for the human RNasP gene, and the target sequence of the primers was artificially connected to the pUC57 vector to form a standard recombinant plasmid; the above pair of primer probes was designed for the Escherichia coli ybbJ gene, and the target sequence of the primers was artificially connected to the pUC57 vector to form a standard recombinant plasmid; the above pair of primer probes was designed for the Klebsiella pneumoniae mgrB gene, and the target sequence of the primers was artificially connected to the pUC57 vector to form a standard recombinant plasmid; the above pair of primer probes was designed for the Staphylococcus aureus Ms The above pair of primer probes were designed for the rB gene, and the target sequence of the primers was artificially connected to the pUC57 vector to form a standard recombinant plasmid; the above pair of primer probes were designed for the PG-mutase gene of Streptococcus agalactiae, and the target sequence of the primers was artificially connected to the pUC57 vector to form a standard recombinant plasmid; the above pair of primer probes were designed for the IS3 gene of beta-hemolytic Streptococcus, and the target sequence of the primers was artificially connected to the pUC57 vector to form a standard recombinant plasmid; the above pair of primer probes were designed for the 16S Burkholderia The above pair of primer probes were designed for the rRNA gene, and the target sequence of the primers was artificially connected to the pUC57 vector to form a standard recombinant plasmid; the above pair of primer probes were designed for the SpaA gene of Clostridium perfringens, and the target sequence of the primers was artificially connected to the pUC57 vector to form a standard recombinant plasmid; the above pair of primer probes were designed for the secA1 gene of Nocardia sp., and the target sequence of the primers was artificially connected to the pUC57 vector to form a standard recombinant plasmid; the above pair of primer probes were designed for the extended-spectrum β-lactamase resistance gene TEM, and the target sequence of the primers was artificially connected to the pUC57 vector to form a standard recombinant plasmid; the above pair of primer probes were designed for the extended-spectrum β-lactamase resistance gene SHV, and the target sequence of the primers was artificially connected to the pUC57 vector to form a standard recombinant plasmid; The above-mentioned pair of primer probes was designed for the beta-lactamase resistance gene CTX-M-9, and the target sequence of the primers was artificially connected to the pUC57 vector to form a standard recombinant plasmid; the above-mentioned pair of primer probes was designed for the methicillin resistance gene mecA, and the target sequence of the primers was artificially connected to the pUC57 vector to form a standard recombinant plasmid; the above-mentioned pair of primer probes was designed for the carbapenemase resistance gene KPC-2, and the target sequence of the primers was artificially connected to the pUC57 vector to form a standard recombinant plasmid; the above-mentioned pair of primer probes was designed for the vancomycin resistance gene VanA, and the target sequence of the primers was artificially connected to the pUC57 vector to form a standard recombinant plasmid; the above-mentioned pair of primer probes was designed for the vancomycin resistance gene VanB, and the target sequence of the primers was artificially connected to the pUC57 vector to form a standard recombinant plasmid;The standard recombinant plasmid powder was resuspended in TE buffer and the concentration was determined by Qubit and diluted to 1.0 × 10; 7 ~1.0×10 3 copies / mL, stored at -80℃ for future use.

[0104] The above primers are shown in Table 1.

[0105] S2. Using the standard recombinant plasmid as a template, the aforementioned kit of this embodiment was used to perform qPCR amplification reaction, wherein the gradient concentration of the standard recombinant plasmid was 1.0×10 7 ~1.0×10 3 copies / mL, the 25μL reaction system for qPCR amplification reaction is:

[0106] Table 2 qPCR amplification reaction system

[0107]

[0108]

[0109] Table 3 Fluorescence amplification instrument reaction program

[0110]

[0111] Signals were collected at the corresponding time, and the Ct value and Tm value of the qPCR product were obtained at the end of the reaction.

[0112] S3. After qPCR amplification reaction and collecting fluorescence signals according to the above method, the Tm value of the qPCR product is obtained. When the Tm value of the FAM channel is 72±1, it means that human genes are detected in the biological sample and the sample sampling is qualified; when the Tm value of the FAM channel is 65±1, it means that Escherichia coli is contained in the biological sample;

[0113] When the Tm value of the FAM channel is 70 ± 1, it means that the biological sample contains Klebsiella pneumoniae;

[0114] When the Tm value of the VIC channel is 62 ± 1, it means that the biological sample contains Staphylococcus aureus;

[0115] When the Tm value of the VIC channel is 68 ± 1, it means that the biological sample contains Streptococcus agalactiae;

[0116] When the Tm value of the VIC channel is 72±1, it means that the biological sample contains beta-hemolytic streptococci;

[0117] When the ROX channel Tm value is 64 ± 1, it means that the biological sample contains Burkholderia;

[0118] When the ROX channel Tm value is 68 ± 1, it means that the biological sample contains Clostridium perfringens;

[0119] When the ROX channel Tm value is 70 ± 1, it means that the biological sample contains Nocardia;

[0120] When the Tm value of the CY5 channel is 70±1, it means that the biological sample is resistant to extended-spectrum β-lactamases;

[0121] When the Tm value of the CY5 channel is 63 ± 1, it means that the biological sample is resistant to methicillin;

[0122] When the Tm value of the CY5 channel is 59 ± 1, it means that the biological sample is resistant to carbapenemase;

[0123] When the Tm value of the CY5 channel is 66±1, it means that the biological sample is resistant to vancomycin.

[0124] Subsequently, the type of pathogen and drug resistance in the sample are determined according to the Tm value of the fusion curve diagram of the above different channels.

[0125] S4. On the basis of the above, a standard curve is established through the Ct value of the standard recombinant plasmid and each gradient concentration, and the Ct value of the sample to be tested is substituted into the known standard curve to calculate the content of the sample to be tested.

[0126] 2. Experimental results and analysis

[0127] (1) Determination and analysis of Tm value

[0128] Human genes 1.0×10 7 ~1.0×10 3 The recombinant plasmid was used as template for amplification to obtain the FAM channel peak with a Tm value of 71.7-72.3. 7 ~1.0×10 3 The recombinant plasmid was used as a template for amplification to obtain the FAM channel peak, with a Tm value of 64.7-64.9. 7 ~1.0×10 3 The recombinant plasmid was used as a template for amplification to obtain the FAM channel peak, with a Tm value of 69.4-69.6. 7 ~1.0×10 3 The recombinant plasmid was used as a template for amplification to obtain the VIC channel peak, with a Tm value of 61.7-62.0. 7 ~1.0×10 3The recombinant plasmid was used as a template for amplification, and the VIC channel peak was obtained, with a Tm value of 68.4-68.5. 7 ~1.0×10 3 The recombinant plasmid was used as a template for amplification to obtain the VIC channel peak, with a Tm value of 71.9-72.4. 7 ~1.0×10 3 The recombinant plasmid was used as a template for amplification to obtain the ROX channel peak, with a Tm value of 63.7-64.5. 7 ~1.0×10 3 The recombinant plasmid was used as a template for amplification to obtain the ROX channel peak, with a Tm value of 67.9-68.4. 7 ~1.0×10 3 The recombinant plasmid was used as a template for amplification to obtain the ROX channel peak, with a Tm value of 70.4-70.8; the extended-spectrum β-lactamase resistance 1.0×10 7 ~1.0×10 3 The recombinant plasmid was used as a template for amplification to obtain the CY5 channel peak, with a Tm value of 69.5-70.7; the methicillin-resistant 1.0×10 7 ~1.0×10 3 The recombinant plasmid was used as a template for amplification, and the CY5 channel peak was obtained, with a Tm value of 62.4-63.1; the carbapenemase resistance of 1.0×10 7 ~1.0×10 3 The recombinant plasmid was used as a template for amplification to obtain the CY5 channel peak, with a Tm value of 59.1-59.4. 7 ~1.0×10 3 The recombinant plasmid with 100 copies / mL was used as a template for amplification, and the CY5 channel peak was obtained, with a Tm value of 64.1-64.8.

[0129] The specific test results of each channel are shown in the following table:

[0130] Table 4 Tm values ​​of FAM channels

[0131]

[0132] Table 5 Tm values ​​of VIC channels

[0133]

[0134] Table 6 Tm values ​​of ROX channel

[0135]

[0136]

[0137] Table 7 Tm values ​​of CY5 channel

[0138]

[0139] (2) Establishment of standard curve

[0140] 1) Escherichia coli 1.0×10 7 ~1.0×10 3 The recombinant plasmid with 0.1% copies / mL was used as the template. The linear regression equation of the standard curve was Ct=-3.239Log(Conc)+43.910, and the amplification efficiency was 103.601%, R 2 =0.999 means that in 1.0×10 7 ~1.0×10 3 There is a good linear relationship in the range of copies / mL, and the minimum detectable 3 copies / mL recombinant plasmid;

[0141] 2) Klebsiella pneumoniae 1.0×10 7 ~1.0×10 3 The recombinant plasmid with 0.1% copies / mL was used as the template. The linear regression equation of the standard curve was Ct=-3.278Log(Conc)+44.709, and the amplification efficiency was 101.864%, R 2 =0.999 means that in 1.0×10 7 ~1.0×10 3 There is a good linear relationship in the range of copies / mL, and the minimum detectable 3 copies / mL recombinant plasmid;

[0142] 3) Staphylococcus aureus 1.0×10 7 ~1.0×10 3 The standard curve was established with the recombinant plasmid of 1000 copies / mL as the template. The linear regression equation was Ct=-3.336Log(Conc)+45.064, and the amplification efficiency was 99.424%. 2 =1.000 means that in 1.0×10 7 ~1.0×10 3 There is a good linear relationship in the range of copies / mL, and the minimum detectable 3copies / mL recombinant plasmid;

[0143] 4) 1.0×10 Streptococcus agalactiae 7 ~1.0×10 3 The standard curve was established with the recombinant plasmid of 1000 copies / mL as the template. The linear regression equation was Ct=-3.261Log(Conc)+44.638, and the amplification efficiency was 102.606%, R 2 =0.999 means that in 1.0×10 7 ~1.0×10 3 There is a good linear relationship in the range of copies / mL, and the minimum detectable 3 copies / mL recombinant plasmid;

[0144] 5) 1.0×10 beta-hemolytic streptococci 7 ~1.0×10 3 The standard curve was established with the recombinant plasmid of 1000 copies / mL as the template. The linear regression equation was Ct=-3.153Log(Conc)+44.599, and the amplification efficiency was 107.561%. 2 =0.999 means that in 1.0×10 7 ~1.0×10 3 There is a good linear relationship in the range of copies / mL, and the minimum detectable 3 copies / mL recombinant plasmid;

[0145] 6) Burkholderia 1.0×10 7 ~1.0×10 3 The recombinant plasmid with 0.1% copies / mL was used as the template. The linear regression equation of the standard curve was Ct=-3.153Log(Conc)+44.850, and the amplification efficiency was 107.568%, R 2 =0.998 means that in 1.0×10 7 ~1.0×10 3 There is a good linear relationship in the range of copies / mL, and the minimum detectable 3 copies / mL recombinant plasmid;

[0146] 7) Clostridium perfringens 1.0×10 7 ~1.0×10 3 The standard curve was established with the recombinant plasmid of 1000 copies / mL as the template. The linear regression equation was Ct=-3.261Log(Conc)+44.630, and the amplification efficiency was 102.615%, R 2 =0.998 means that in 1.0×107 ~1.0×10 3 There is a good linear relationship in the range of copies / mL, and the minimum detectable 3 copies / mL recombinant plasmid;

[0147] 8) Nocardia 1.0×10 7 ~1.0×10 3 The standard curve was established with the recombinant plasmid of 1000 copies / mL as the template. The linear regression equation was Ct=-3.336Log(Conc)+45.046, and the amplification efficiency was 99.424%. 2 =1.000 means that in 1.0×10 7 ~1.0×10 3 There is a good linear relationship in the range of copies / mL, and the minimum detectable 3 copies / mL recombinant plasmid;

[0148] 9) Extended-spectrum β-lactamase TEM resistance gene 1.0×10 7 ~1.0×10 3 The standard curve was established with the recombinant plasmid of 10000 copies / mL as the template. The linear regression equation was Ct=-3.311Log(Conc)+44.188, and the amplification efficiency was 100.473%. 2 =1.000 means that in 1.0×10 7 ~1.0×10 3 There is a good linear relationship in the range of copies / mL, and the minimum detectable 3 copies / mL recombinant plasmid;

[0149] 10) Extended-spectrum β-lactamase SHV resistance gene 1.0×10 7 ~1.0×10 3 The standard curve was established with the recombinant plasmid of 1000 copies / mL as the template. The linear regression equation was Ct=-3.262Log(Conc)+43.523, and the amplification efficiency was 102.576%. 2 =0.999 means that in 1.0×10 7 ~1.0×10 3 There is a good linear relationship in the range of copies / mL, and the minimum detectable 3 copies / mL recombinant plasmid;

[0150] 11) Extended-spectrum β-lactamase CTX-M-9 resistance gene 1.0×10 7 ~1.0×103 The standard curve was established with the recombinant plasmid of 1000 copies / mL as the template. The linear regression equation was Ct=-3.286Log(Conc)+43.487, and the amplification efficiency was 101.508%, R 2 =1.000 means that in 1.0×10 7 ~1.0×10 3 There is a good linear relationship in the range of copies / mL, and the minimum detectable 3 copies / mL recombinant plasmid;

[0151] 12) Methicillin mecA resistance gene 1.0×10 7 ~1.0×10 3 The standard curve was established with 0.0000 copies / mL recombinant plasmid as template. The linear regression equation was Ct=-3.315Log(Conc)+37.871, and the amplification efficiency was 100.310%, R 2 =1.000 means that in 1.0×10 7 ~1.0×10 3 There is a good linear relationship in the range of copies / mL, and the minimum detectable 3 copies / mL recombinant plasmid;

[0152] 13) Carbapenemase KPC-2 resistance gene 1.0×10 7 ~1.0×10 3 The standard curve was established with the recombinant plasmid of 1000 copies / mL as the template. The linear regression equation was Ct=-3.376Log(Conc)+43.810, and the amplification efficiency was 97.801%. 2 =1.000 means that in 1.0×10 7 ~1.0×10 3 There is a good linear relationship in the range of copies / mL, and the minimum detectable 3 copies / mL recombinant plasmid;

[0153] 14) Vancomycin resistance gene 1.0×10 7 ~1.0×10 3 The standard curve was established with the recombinant plasmid of 1000 copies / mL as the template. The linear regression equation was Ct=-3.463Log(Conc)+45.997, and the amplification efficiency was 94.429%. 2 =1.000 means that in 1.0×10 7 ~1.0×10 3There is a good linear relationship in the range of copies / mL, and the minimum detectable 3 copies / mL recombinant plasmid;

[0154] 15) Vancomycin resistance gene 1.0×10 7 ~1.0×10 3 The recombinant plasmid with 0.1% copies / mL was used as the template. The linear regression equation of the standard curve was Ct=-3.592Log(Conc)+46.170, and the amplification efficiency was 89.840%, R 2 =0.999 means that in 1.0×10 7 ~1.0×10 3 There is a good linear relationship in the range of copies / mL, and the minimum detectable 3 copies / mL recombinant plasmid. The results are shown in Figures 5 to 12 .

[0155] It can be seen that the kit of the present application can detect a minimum of 1.0×10 3 copies / mL of 8 pathogens and 7 recombinant plasmids of drug-resistant genes.

[0156] Example 2 Determination of the detection limit of the kit

[0157] 1. Experimental methods

[0158] 500 copies / mL of Escherichia coli, 500 copies / mL of Klebsiella pneumoniae, 500 copies / mL of Staphylococcus aureus, 500 copies / mL of Streptococcus agalactiae, 500 copies / mL of beta-hemolytic Streptococcus, 1.0×10 3 copies / mL Burkholderia, 500 copies / mL Clostridium perfringens, 1.0×10 3 copies / mL Nocardia, 1.0×10 3 copies / mL of extended-spectrum β-lactamase, 500 copies / mL of methicillin, 500 copies / mL of carbapenemase, and 1.0×10 3 The recombinant plasmid of copies / mL vancomycin standard was used as a template for qPCR amplification reaction, and the operation method and conditions were referred to Example 1. The reaction was repeated 10 times, and the positive detection rate and CV value were calculated to evaluate the detection limit of the method.

[0159] 2. Experimental results and analysis

[0160] From the results in Table 8, it can be seen that the test kit of the present application has high detection efficiency, with a positive detection rate of 100% for each pathogen, and high sensitivity. The CV values ​​calculated according to the Tm values ​​do not exceed 5%. The concentration of each pathogen in the sample and its drug resistance can be accurately determined, which is convenient for providing more targeted and effective treatment plans in the future.

[0161] Table 8 Detection rate and CV value determination results

[0162]

[0163]

[0164]

[0165]

[0166] Example 3 Determination of the specificity of the kit

[0167] 1. Experimental methods

[0168] The nucleic acids in samples of Salmonella, Shigella, Acinetobacter baumannii, Haemophilus, Pseudomonas aeruginosa, Proteus, Streptococcus pneumoniae, Staphylococcus epidermidis, etc. were used as templates for qPCR amplification reaction, wherein the sample concentration was 1.0×10 5 The operation method and conditions were as in Example 1. The specificity of the method was evaluated.

[0169] 2. Experimental results and analysis

[0170] Test results such as Fig.13 From left to right, the blue lines are Escherichia coli and Klebsiella pneumoniae, the green lines are Staphylococcus aureus, Streptococcus agalactiae, and beta-hemolytic Streptococcus, the red lines are Burkholderia, Clostridium perfringens, and Nocardia, and the yellow lines are Salmonella, Shigella, Acinetobacter baumannii, Haemophilus, Pseudomonas aeruginosa, Proteus, Streptococcus pneumoniae, and Staphylococcus epidermidis. None of them have melting curve peaks.

[0171] It has been verified that the results of this method for detecting Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus agalactiae, beta-hemolytic Streptococcus, Burkholderia, Clostridium perfringens, and Nocardia were all positive, and the results of detecting other related pathogens were all negative, indicating that the kit and detection method have good specificity.

[0172] Example 4 Detection of real samples of pathogens related to skin and soft tissue infection by this kit

[0173] 1. Experimental methods

[0174] (1) Sample pretreatment

[0175] The kit of Example 1 of the present application was used to detect the type, concentration and drug resistance of pathogens related to skin and soft tissue infections on 96 real samples (21 Escherichia coli samples, 16 Klebsiella pneumoniae samples, 18 Staphylococcus aureus samples, 7 Streptococcus agalactiae samples, 13 beta-hemolytic Streptococcus samples, 6 Burkholderia samples, 10 Clostridium perfringens samples and 5 Nocardia samples), and the specific method is as follows:

[0176] Use a swab to wipe the skin and soft tissue of the infected area of ​​the SSTI patient, place the sampled swab in a sample release agent, vortex mix for 30 seconds, and let it stand at room temperature for 5 minutes. The supernatant is added to the qPCR reaction solution as an amplification template for the qPCR experiment for amplification. After the reaction, the Tm value and Ct value of the qPCR product are obtained. The pathogen and whether it is drug-resistant are determined based on the fluorescence channel and Tm value, and the Ct value is substituted into the linear regression equation of the corresponding pathogen to calculate the pathogen concentration.

[0177] The sample release agent is composed of the following components: 2% by volume of Tween 20, 5% by volume of Triton X-100, 2mM of guanidine isothiocyanate, 10mM of tris(hydroxymethyl)aminomethane salt and 1mM of disodium ethylenediaminetetraacetate, and the balance is sterile water. The pH of the nucleic acid release agent provided by the present invention is 7.4±0.2.

[0178] 2. Experimental results and analysis

[0179] The test results are shown in Table 9. From the results, it can be seen that the detection compliance rate of 96 real samples using this kit reached 100%, the detection sensitivity was high, and the drug resistance and concentration of each pathogen in the sample could be accurately determined, as shown in Table 9 for details.

[0180] Table 9 Test results of samples

[0181]

[0182]

[0183]

[0184]

[0185] Example 5 Performance comparison of comparative kits with different primer-probe combinations

[0186] 1. Experimental methods

[0187] This experiment provides kits of Comparative Example 1 and Comparative Example 2. The difference between the kit of Comparative Example 1 and the kit of Example 1 is that the primer probe compositions used for the VIC channel targets Staphylococcus aureus, Streptococcus agalactiae and beta-hemolytic Streptococcus are different, and the difference between the kit of Comparative Example 2 and the kit of Example 1 is that the primer probe compositions used for the ROX channel targets Clostridium perfringens and Nocardia are different, as shown in Table 10. The fluorescent groups on each primer probe of each control group refer to Example 1 for comparative analysis, and the preparation method of the kit is the same as that of the aforementioned example.

[0188] Table 10 Primer probe sequences of the control group

[0189]

[0190] The kit prepared in Example 1 of the present application, the kit prepared in Comparative Example 1 and Comparative Example 2 were used to test Staphylococcus aureus, Streptococcus agalactiae, beta-hemolytic Streptococcus, Burkholderia, Clostridium perfringens and Nocardia 1.0×10 4 copies / mL and 1.0×10 3 The experimental results of the kits prepared in Comparative Example 1 and Comparative Example 2 are as follows: Fig.14 , 15 shown.

[0191] 2. Experimental results and analysis

[0192] Compared with Example 1, in Comparative Example 1, there is no melting peak for Staphylococcus aureus, and the melting peaks for Streptococcus agalactiae and beta-hemolytic Streptococcus are low, and only 1.0×10 4 The recombinant plasmid of Streptococcus agalactiae can read the peak value, that is, Streptococcus agalactiae can only detect 1.0×10 4 copies / mL recombinant plasmid, 1.0×10 3 copies / mL, Staphylococcus aureus and beta-hemolytic Streptococcus were not detected at 1.0×10 4 copies / mL and 1.0×10 3 copies / mL recombinant plasmid.

[0193] Compared with Example 1, there was no melting peak for Clostridium perfringens in Comparative Example 2, and only 1.0×10 4 copies / mL recombinant plasmid can read the melting peak, 1.0×10 3 The melting peak of the recombinant plasmid could not be read, that is, Clostridium perfringens could not be detected at 1.0×10 4 copies / mL and 1.0×10 3copies / mL of recombinant plasmid, and only 1.0×10 4 copies / mL recombinant plasmid, 1.0×10 3 copies / mL, and under the influence of other primer-probe combinations, the Burkholderia with the same primers and probes as in Example 1 could only detect 1.0×10 4 copies / mL recombinant plasmid, 1.0×10 3 copies / mL.

[0194] This example shows that the kit prepared by the present invention has accurate results, high sensitivity and good specificity of primer design. This also shows that the primer probe sequence in the primer probe combination in the kit of the present invention cannot be replaced at will, otherwise its technical effect cannot achieve the expected effect.

[0195] Example 6 Performance comparison of comparative kits with different primer and probe dosages

[0196] 1. Experimental methods

[0197] This experiment provides a kit of comparative example 3. The difference between the kit of comparative example 3 and the kit of example 1 is that the amount of primer probes used for the FAM channel Escherichia coli, Klebsiella pneumoniae, and human internal reference is different, as shown in Table 11. The fluorescent groups on each primer probe of each control group refer to Example 1 for comparative analysis, and the preparation method of the kit is the same as that of the aforementioned example.

[0198] Table 11 qPCR amplification reaction system of comparative example 3

[0199]

[0200]

[0201] The kit prepared in Example 1 of the present application and the kit prepared in Comparative Example 3 were respectively used to test Escherichia coli, Klebsiella pneumoniae, and human internal reference 1.0×10 4 copies / mL and 1.0×10 3 The experimental results of the kit prepared in Example 3 are as follows: Fig.16 shown.

[0202] 2. Experimental results and analysis

[0203] Compared with Example 1, the human internal reference in Comparative Example 3 was 1.0×10 4 copies / mL and 1.0×10 3copies / mL recombinant plasmids can be detected, but the melting peak is too high, the melting peak of Escherichia coli and Klebsiella pneumoniae is low, and the melting peak of Escherichia coli and Klebsiella pneumoniae is only 1.0×10 4 The peak value can be read by the recombinant plasmid, that is, Escherichia coli and Klebsiella pneumoniae can only be detected at 1.0×10 4 copies / mL recombinant plasmid, 1.0×10 3 copies / mL, the detection sensitivity is not as good as that of Example 1.

[0204] This example shows that the kit prepared by the present invention has accurate results, high sensitivity and good specificity of the reaction system. This also shows that the amount of primers and probes in the primer-probe combination in the kit of the present invention cannot be modified at will, otherwise the technical effect cannot achieve the expected effect.

[0205] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and concept of the present invention, and all these changes or substitutions should fall within the protection scope of the claims attached to the present invention.

Claims

1. A primer-probe combination for simultaneously detecting pathogens related to skin and soft tissue infections and their drug resistance genes, It is characterized in that The invention comprises a primer-probe combination targeting the ybbJ gene of Escherichia coli, the mgrB gene of Klebsiella pneumoniae, the MsrB gene of Staphylococcus aureus, the PG-mutase gene of Streptococcus agalactiae, the IS3 gene of beta-hemolytic Streptococcus, the 16S rRNA gene of Burkholderia, the SpaA gene of Clostridium perfringens, the secA1 gene of Nocardia, and the extended-spectrum β-lactamase resistance gene, the methicillin resistance gene, the carbapenemase resistance gene and the vancomycin resistance gene. The primer probe for detecting the ybbJ gene of Escherichia coli includes: an upstream primer ybbJ-F having a sequence as shown in SEQ ID NO.1, a downstream primer ybbJ-R having a sequence as shown in SEQ ID NO.2, and a fluorescent probe ybbJ-P having a sequence as shown in SEQ ID NO.3; The primer probe for detecting mgrB gene of Klebsiella pneumoniae includes: an upstream primer mgrB-F having a sequence as shown in SEQ ID NO.4, a downstream primer mgrB-R having a sequence as shown in SEQ ID NO.5, and a fluorescent probe mgrB-P having a sequence as shown in SEQ ID NO.6; The primer probe for detecting the MsrB gene of Staphylococcus aureus includes: an upstream primer MsrB-F whose sequence is shown in SEQ ID NO.7, a downstream primer MsrB-R whose sequence is shown in SEQ ID NO.8, and a fluorescent probe MsrB-P whose sequence is shown in SEQ ID NO.9; The primer probe for detecting the PG-mutase gene of Streptococcus agalactiae comprises: an upstream primer PG-mutase-F having a sequence as shown in SEQ ID NO.10, a downstream primer PG-mutase-R having a sequence as shown in SEQ ID NO.11, and a fluorescent probe PG-mutase-P having a sequence as shown in SEQ ID NO.12; The primer probe for detecting IS3 gene of beta-hemolytic streptococcus includes: an upstream primer IS3-F whose sequence is shown in SEQ ID NO.13, a downstream primer IS3-R whose sequence is shown in SEQ ID NO.14, and a fluorescent probe IS3-P whose sequence is shown in SEQ ID NO.15; The primer probe for detecting the 16S rRNA gene of Burkholderia includes: an upstream primer 16S rRNA-F having a sequence as shown in SEQ ID NO.16, a downstream primer 16S rRNA-F having a sequence as shown in SEQ ID NO.17, and a fluorescent probe 16S rRNA-P having a sequence as shown in SEQ ID NO.18; The primer probe for detecting the SpaA gene of Clostridium perfringens includes: an upstream primer SpaA-F having a sequence as shown in SEQ ID NO.19, a downstream primer SpaA-R having a sequence as shown in SEQ ID NO.20, and a fluorescent probe SpaA-P having a sequence as shown in SEQ ID NO.21; The primer probe for detecting the Nocardia secA1 gene includes: an upstream primer secA1-F having a sequence as shown in SEQ ID NO.22, a downstream primer secA1-R having a sequence as shown in SEQ ID NO.23, and a fluorescent probe secA1-P having a sequence as shown in SEQ ID NO.24; The primer probe for detecting the β-lactamase resistance gene TEM includes: an upstream primer TEM-F whose sequence is shown in SEQ ID NO.25, a downstream primer TEM-R whose sequence is shown in SEQ ID NO.26, and a fluorescent probe TEM-P whose sequence is shown in SEQ ID NO.27; The primer probe for detecting the β-lactamase resistance gene SHV includes: an upstream primer SHV-F whose sequence is shown in SEQ ID NO.28, a downstream primer SHV-R whose sequence is shown in SEQ ID NO.29, and a fluorescent probe SHV-P whose sequence is shown in SEQ ID NO.30; The primer probe for detecting the β-lactamase resistance gene CTX-M-9 includes: an upstream primer CTX-M-9-F having a sequence as shown in SEQ ID NO.31, a downstream primer CTX-M-9-R having a sequence as shown in SEQ ID NO.32, and a fluorescent probe CTX-M-9-P having a sequence as shown in SEQ ID NO.33; The primer probe for detecting the methicillin resistance gene mecA comprises: an upstream primer mecA-F having a sequence as shown in SEQ ID NO.34, a downstream primer mecA-R having a sequence as shown in SEQ ID NO.35, and a fluorescent probe mecA-P having a sequence as shown in SEQ ID NO.36; The primer probe for detecting the carbapenemase resistance gene KPC-2 comprises: an upstream primer KPC-2-F having a sequence as shown in SEQ ID NO.37, a downstream primer KPC-2-R having a sequence as shown in SEQ ID NO.38, and a fluorescent probe KPC-2-P having a sequence as shown in SEQ ID NO.38; The primer probe for detecting the vancomycin resistance gene VanA includes: an upstream primer VanA-F whose sequence is shown in SEQ ID NO.40, a downstream primer VanA-R whose sequence is shown in SEQ ID NO.41, and a fluorescent probe VanA-P whose sequence is shown in SEQ ID NO.42; The primer probe for detecting the vancomycin resistance gene VanB includes: an upstream primer VanB-F whose sequence is shown in SEQ ID NO.43, a downstream primer VanB-R whose sequence is shown in SEQ ID NO.44, and a fluorescent probe VanB-P whose sequence is shown in SEQ ID NO.45; The 5' end of the fluorescent probe is labeled with a fluorescent reporter group, and the 3' end of the fluorescent probe is labeled with a fluorescent quencher group.

2. The primer-probe combination according to claim 1, It is characterized in that It also includes primer probes for detecting the internal reference RNaseP gene: an upstream primer RNaseP-F1 with a sequence as shown in SEQ ID NO.46, a downstream primer RNaseP-R1 with a sequence as shown in SEQ ID NO.47, and a fluorescent probe RNaseP-P1 with a sequence as shown in SEQ ID NO.

48.

3. The primer-probe combination according to claim 1 or 2, It is characterized in that The fluorescent reporter group is selected from FAM, ROX, CY5, and VIC; the fluorescent quencher group is selected from BHQ1, BHQ2, and BHQ3.

4. A kit for simultaneously detecting pathogens related to skin and soft tissue infections and their drug resistance genes, It is characterized in that Comprising the primer-probe combination as claimed in claim 1.

5. The kit according to claim 4, It is characterized in that Also includes: DNA polymerase, PCR reaction buffer.

6. The kit according to claim 5, It is characterized in that The kit also includes: a sample release agent; the sample release agent includes the following components: 2% by volume of Tween 20, 5% by volume of Triton X-100, 2mM guanidine isothiocyanate, 10mM tris(hydroxymethyl)aminomethane salt and 1mM disodium ethylenediaminetetraacetic acid, and the remainder is sterile water; the pH is 7.4±0.

2.

7. Use of the primer-probe combination according to claim 1 in preparing a kit for detecting pathogens related to skin and soft tissue infections and their drug resistance genes.

8. A method for simultaneously detecting pathogens related to skin and soft tissue infections and their drug resistance genes. It is characterized in that The following steps are involved: S1, pre-treating the collected sample to be tested and adding it to a PCR reaction tube, wherein the PCR reaction tube also contains the primer-probe combination as claimed in claim 1 and a PCR reaction solution, and performing qPCR amplification after mixing; after the reaction is completed, collecting the fluorescence signal of each channel to obtain the Tm value of the melting curve of each channel; S2. Determine whether the sample to be tested contains each pathogen based on the Tm value of the melting curve of each channel in the PCR reaction system. Optionally, the specific analysis and judgment method of step S2 is: When the Tm value of the FAM channel is 65 ± 1, it means that the biological sample contains E. coli; When the Tm value of the FAM channel is 70 ± 1, it means that the biological sample contains Klebsiella pneumoniae; When the Tm value of the VIC channel is 62 ± 1, it means that the biological sample contains Staphylococcus aureus; When the Tm value of the VIC channel is 68 ± 1, it means that the biological sample contains Streptococcus agalactiae; When the Tm value of the VIC channel is 72±1, it means that the biological sample contains beta-hemolytic streptococci; When the ROX channel Tm value is 64 ± 1, it means that the biological sample contains Burkholderia; When the ROX channel Tm value is 68 ± 1, it means that the biological sample contains Clostridium perfringens; When the ROX channel Tm value is 70 ± 1, it means that the biological sample contains Nocardia; When the Tm value of the CY5 channel is 70±1, it means that the biological sample is resistant to extended-spectrum β-lactamases; When the Tm value of the CY5 channel is 63 ± 1, it means that the biological sample is resistant to methicillin; When the Tm value of the CY5 channel is 59 ± 1, it means that the biological sample is resistant to carbapenemase; When the Tm value of the CY5 channel is 66±1, it means that the biological sample is resistant to vancomycin.

9. The method according to claim 9, It is characterized in that The following steps are also included: S3. Based on the standard curve of the standard recombinant plasmid of each target gene, the concentration of the target pathogen in the sample to be tested can be obtained according to the fluorescence amplification Ct value of the sample to be tested.

10. The method according to claim 8, It is characterized in that In the step S1, the pretreatment method of the sample to be tested is: use a swab to wipe the infected skin and soft tissue of a patient with skin and soft tissue infection, put the sampled swab into a sample release agent, vortex mix, and stand at room temperature. The supernatant can be used as an amplification template for the PCR experiment.

Images