Primer and probe for rapidly detecting bone and joint infection pathogens and detection method

Abstract

The invention relates to a primer, a probe and a detection method for rapidly detecting bone and joint infection pathogens. Comprising at least three groups of primers and probes in primers and probes respectively aiming at staphylococcus aureus, acinetobacter baumannii, staphylococcus epidermidis, staphylococcus hemolyticus, pseudomonas aeruginosa, enterobacter cloacae, escherichia coli, methicillin-resistant staphylococcus aureus and streptococcus pyogenes. According to the invention, the defects of small flux and few types of detectable pathogens in the existing bone and joint infection detection method are overcome, and 9 groups of specially designed primers and probes respectively aim at 9 bone and joint infection pathogens covering 90% or more of bone and joint infection cases; the kit has the advantages that the flux is large (at least three bone and joint infection pathogens can be detected at a time), multiple types of pathogens can be detected, and the specificity and the sensitivity are good.

Description

Technical Field

[0001] The invention relates to a primer and a probe for rapid detection of pathogens of bone and joint infection and a detection method, and is applied in the field of rapid detection of pathogens of bone and joint infection. Background Art

[0002] Bone and joint infection refers to infection caused by pathogens invading bone tissue or joint cavity. It is common in clinical practice and difficult to treat. Bone and joint infection mainly includes hematogenous osteomyelitis, traumatic osteomyelitis, infectious arthritis, and infection after implantation or artificial joint replacement. Due to the hidden lesions of bone and joint infection, it is difficult to obtain etiological results, which makes it difficult to diagnose and treat. It is one of the difficult diseases faced by clinicians and patients. Complications such as inflammatory pain, bone defects, and functional disorders caused by bone and joint infection greatly affect the quality of life of patients. In addition, the long treatment time, adverse drug reactions, and high surgical costs often bring great family pressure and social burden to patients. The key to the treatment of bone and joint infection is to formulate appropriate antibiotic treatment plans based on accurate and rapid etiological diagnosis, which is crucial to shorten the course of infection, reduce complications, and even increase the cure rate. Due to the unclear diagnosis of pathogens, strong broad-spectrum antibiotics can only be used to cover all common pathogens for anti-infection treatment, which greatly increases the treatment time and cost, and also brings more adverse prognostic effects. Therefore, there is an urgent need for a method that is highly sensitive, time-saving, and can be used for immediate diagnosis of bone and joint infections (POCT) at the bedside and during surgery.

[0003] With the continuous development of molecular diagnostic methods, molecular diagnostic technology based on multiple polymerase chain reaction (PCR) has largely made up for the defects of traditional bacterial culture and immunological detection methods for the detection of bone and joint infection pathogens, making the detection of bone and joint infection pathogens faster, and improving sensitivity and specificity. For the screening and detection of bacteria, traditional fluorescent quantitative PCR can obtain results within 3-4 hours, with good specificity and sensitivity, but its throughput is small, and it can only detect one or two bacteria at a time, and it needs to configure a series of gradient reaction systems for detection. However, there are many types of bone and joint infection pathogens, and the application value of fluorescent quantitative PCR is limited when large-scale rapid detection is required. Some previous invention patents (CN201810514985.8) also have designs for the detection of bone and joint infection pathogens, but the target pathogens are few, cannot cover most of the bone and joint infection pathogens, and are targeted at children's bone and joint infections, and cannot be promoted to the general public of all ages. According to the statistical results of relevant research by scholars at home and abroad, the most common pathogens of bone and joint infection are: Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hemolyticus, methicillin-resistant Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa, Escherichia coli, Enterobacter cloacae, Acinetobacter baumannii, etc. These pathogens can cover more than 90% of bone and joint infection cases. We use molecular diagnostic technology to quickly and accurately detect the above pathogens at the same time, which is of great help to the clinical diagnosis and treatment of bone and joint infection.

[0004] Therefore, it has become an urgent task to provide primers and probes as well as a detection method for rapid detection of bone and joint infection pathogens, which have high throughput (capable of detecting at least 3 bone and joint infection pathogens at a time), can detect a wide variety of pathogens, have good specificity and sensitivity, and can directly detect samples without extracting sample nucleic acid. Summary of the invention

[0005] In order to overcome the shortcomings of existing bone and joint infection detection methods such as low throughput and a small number of detectable pathogens, the present invention provides primers and probes for rapid detection of bone and joint infection pathogens and a detection method. Through specially designed 9 sets of primers and probes respectively targeting 9 types of bone and joint infection pathogens covering more than 90% of bone and joint infection cases, the method has the advantages of high throughput (capable of detecting at least 3 types of bone and joint infection pathogens at a time), a large number of detectable pathogens, and good specificity and sensitivity.

[0006] The technical solution of the present invention is as follows:

[0007] A primer and probe for rapid detection of pathogens of bone and joint infection, comprising at least three sets of primers and probes for Staphylococcus aureus, Acinetobacter baumannii, Staphylococcus epidermidis, Staphylococcus haemolyticus, Pseudomonas aeruginosa, Enterobacter cloacae, Escherichia coli, methicillin-resistant Staphylococcus aureus and Streptococcus pyogenes;

[0008] The sequence of the upstream primer aureusF for Staphylococcus aureus is shown in SEQ ID No: 1, the sequence of the downstream primer aureusR is shown in SEQ ID No: 2, and the sequence of the probe aureusP is shown in SEQ ID No: 3;

[0009] The sequence of the upstream primer baumanniiF for Acinetobacter baumannii is shown in SEQ ID No: 4, the sequence of the downstream primer baumanniiR is shown in SEQ ID No: 5, and the sequence of the probe baumanniiP is shown in SEQ ID No: 6;

[0010] The sequence of the upstream primer epidermidisF for Staphylococcus epidermidis is shown in SEQ ID No: 7, the sequence of the downstream primer epidermidisR is shown in SEQ ID No: 8, and the sequence of the probe epidermidisP is shown in SEQ ID No: 9;

[0011] The sequence of the upstream primer haemolyticusF for hemolytic Staphylococcus is shown in SEQ ID No: 10, the sequence of the downstream primer haemolyticusR is shown in SEQ ID No: 11, and the sequence of the probe haemolyticusP is shown in SEQ ID No: 12;

[0012] The sequence of the upstream primer aeruginosaF for Pseudomonas aeruginosa is shown in SEQ ID No: 13, the sequence of the downstream primer aeruginosaR is shown in SEQ ID No: 14, and the sequence of the probe aeruginosaP is shown in SEQ ID No: 15;

[0013] The sequence of the upstream primer cloacaeF for Enterobacter cloacae is shown in SEQ ID No: 16, the sequence of the downstream primer cloacaeR is shown in SEQ ID No: 17, and the sequence of the probe cloacaeP is shown in SEQ ID No: 18;

[0014] The sequence of the upstream primer coliF for Escherichia coli is shown in SEQ ID No: 19, the sequence of the downstream primer coliR is shown in SEQ ID No: 20, and the sequence of the probe coliP is shown in SEQ ID No: 21;

[0015] The sequence of the upstream primer mecA-F for methicillin-resistant Staphylococcus aureus is shown in SEQ ID No: 22, the sequence of the downstream primer mecA-R is shown in SEQ ID No: 23, and the sequence of the probe mecA-probe is shown in SEQ ID No: 24;

[0016] The sequence of the upstream primer pyogenesF for Streptococcus pyogenes is shown in SEQ ID No:25, the sequence of the downstream primer pyogenesR is shown in SEQ ID No:26, and the sequence of the probe pyogenesP is shown in SEQ ID No:27.

[0017] The primers and probes for rapid detection of bone and joint infection pathogens and the detection method of the present application are specially designed for 9 groups of primers and probes for 9 types of bone and joint infection pathogens covering more than 90% of bone and joint infection cases, and have the advantages of large throughput (capable of detecting at least 3 types of bone and joint infection pathogens at a time), a wide range of pathogens that can be detected, and good specificity and sensitivity. The primers and probes for rapid detection of bone and joint infection pathogens and the detection method are designed with exclusive primers and probes for each type of bone and joint infection pathogen, and have better specificity and higher sensitivity. By detecting whether the PCR reaction product produces fluorescence, it can be determined whether the patient is infected with the corresponding bone and joint pathogen. It can not only be used for the diagnosis of bone and joint infections in children, but can also be extended to adults or all age segments, regardless of anatomical position differences.

[0018] 1. Specific development and testing of primers and probes

[0019] 1. Find the target gene site

[0020] From the ATCC strain library, standard strains of nine common microorganisms in bone and joint infections, including Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, methicillin-resistant Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa, Escherichia coli, Enterobacter cloacae, and Acinetobacter baumannii, were obtained (see Table 1).

[0021] Table 1 Summary of standard strain information

[0022]

[0023]

[0024] According to the relevant documentation provided by the strain library, each strain was revived and cultured in the P2 biosafety laboratory. The specific method of strain revivification culture is as follows: 1. Prepare sterile agarose blood plates, sterile inoculation loops, and standard strain freeze-dried agents; 2. Scrape a small amount of strains with an inoculation loop and streak inoculate on the sterile agarose blood plate, being careful not to scratch the agar. After inoculation, invert it in a 37°C greenhouse and culture it overnight. Observe the culture results the next day; if colonies appear, they can be used for subsequent experiments.

[0025] The whole genome data of the standard strains of the above 9 strains were downloaded from international genome databases such as NCBI. The whole genome sequence of each strain was compared with the pre-downloaded NCBI nt database using a local alignment algorithm on the server cluster of the Fujian Supercomputing Center to find specific sequences on the genome of each strain, and sorted according to the degree of specificity as candidate target gene sites. Suitable candidate target gene site sequences (sequences shown in SEQ ID No: 28-SEQ ID No: 36) were selected, and suitable specific primers and probes were subsequently designed.

[0026] 2. Primer and probe design and identification

[0027] Primer Express 3.0 software was used to design qPCR primers and probes for the conserved region of the specific fragment (target gene site sequence - sequence SEQ ID No: 28-SEQ ID No: 36), and the specificity was preliminarily identified by the BLAST function of NCBI to screen out the best combination of primers and probes.

[0028] A method for rapid detection of pathogens of bone and joint infection mainly comprises the following steps in sequence:

[0029] (i) performing a one-step multiplex RT-qPCR amplification reaction on the sample DNA using at least three sets of primers and probes for Staphylococcus aureus, Acinetobacter baumannii, Staphylococcus epidermidis, Staphylococcus haemolyticus, Pseudomonas aeruginosa, Enterobacter cloacae, Escherichia coli, methicillin-resistant Staphylococcus aureus, and Streptococcus pyogenes;

[0030] The amplification reaction system is as follows:

[0031]

[0032] The final molar concentration of each upstream primer, each downstream primer and each probe is 10 mM, and the sample is tissue fluid of bone tissue, tissue fluid of joint cavity or prosthesis ultrasonic lysate;

[0033] The amplification reaction procedure is as follows:

[0034]

[0035] (ii) Result analysis: Whether the patient is infected with the corresponding bone and joint pathogens is determined by detecting whether the PCR reaction product is amplified.

[0036] The key to treating bone and joint infections is to develop appropriate antibiotic treatment plans based on accurate and rapid etiological diagnosis, which is crucial to shortening the course of infection, reducing complications and even increasing cure rates. Currently, due to the unclear diagnosis of pathogens, we can only use strong and broad-spectrum antibiotics to cover all common pathogens of bone and joint infections, which greatly increases the treatment time and cost of patients and also brings more adverse prognostic effects.

[0037] The existing conventional PCR does not have specific primers for pathogens of bone and joint infection, and the targets detected at the same time are few, and the sensitivity and specificity are still insufficient. The present application discloses a primer and probe for rapid detection of pathogens of bone and joint infection and a detection method thereof, and designs primers and fluorescent probes for 9 common pathogens of bone and joint infection in orthopedic clinics (which can cover more than 90% of bone and joint infection cases), and utilizes the specificity and sensitivity of primers in the multiple PCR reaction process of rapid detection of pathogens of bone and joint infection. Through the complementarity of the DNA template chains at both ends of the upstream and downstream primer target genes, the template DNA sequence can be quickly and effectively amplified, and the fluorescent probe combined with the target gene can display the PCR amplification efficiency and amplification amount in real time. Therefore, it can help clinicians to quickly diagnose pathogens of bone and joint infection, and use non-broad-spectrum antibiotics that are sensitive and specific to pathogens, avoiding the abuse of broad-spectrum antibiotics and the formation of drug-resistant strains, and helping surgeons make surgical decisions and formulate correct surgical treatment plans. This method for rapid detection of pathogens of bone and joint infections uses 9 sets of primers and probes specially designed for 9 common pathogens of bone and joint infections, combined with one-step multiplex RT-qPCR amplification using Taqman fluorescent probe technology. It does not require pre-treatment of samples and can be directly added to the sample (no nucleic acid separation and purification is required to amplify target DNA from the sample, and no gel electrophoresis is required for separation and quantification), which can greatly save detection time; the test results can be obtained within 1 hour, truly achieving "rapid detection"; and because Taqman fluorescent probe technology is used for rapid dual-temperature amplification, accurate quantitative measurement can be performed during PCR amplification.

[0038] The basic principle of RT-qPCR amplification (combined with Taqman fluorescent probe technology) is to add a specific fluorescent probe at the same time as a pair of primers during PCR amplification, and the two ends of the probe are labeled with a reporter fluorescent group and a quencher fluorescent group respectively. Before amplification begins, the probe is completely bound to any single strand of DNA, and the fluorescent signal emitted by the reporter fluorescent group is absorbed by the quencher fluorescent group, and no fluorescent signal can be detected; during PCR amplification, the 5'→3' nuclease activity of the Taq enzyme will enzymatically degrade the probe, separate the reporter fluorescent group and the quencher fluorescent group, and emit fluorescence, and the number of fluorescent molecules cut is proportional to the number of PCR products. Therefore, by detecting whether the PCR reaction product emits fluorescence and the intensity of the fluorescence, the purpose of detecting whether the PCR product is obtained and the amount of PCR product amplification can be achieved. The following Table 2 is a comparison between SYBR Green dye-based and Taqman-based probe technology.

[0039] Table 2 Comparison of SYBR Green dye-based and Taqman-based probe detection technologies

[0040]

[0041] It can be seen that the Taqman-based probe technology is better than the SYBR Green dye-based probe technology.

[0042] After screening out 9 sets of primers and probes designed for pathogens of bone and joint infection, the multiplex PCR system (one-step multiplex RT-qPCR amplification system) was optimized, and finally the optimal amplification reaction system and reaction procedure were obtained as follows:

[0043] The amplification reaction system is as follows:

[0044]

[0045] Among them, the final molar concentrations of the upstream primer, downstream primer, and probe were all 10 mM;

[0046] The amplification reaction procedure is as follows:

[0047]

[0048] Compared with the prior art, the present invention has the following advantages:

[0049] 1) The primers and probes for rapid detection of bone and joint infection pathogens of the present application are specially designed for 9 sets of primers and probes for 9 bone and joint infection pathogens covering more than 90% of bone and joint infection cases, and a one-step multiplex RT-qPCR amplification combined with Taqman fluorescent probe technology is adopted. It has the advantages of high throughput (capable of detecting at least 3 bone and joint infection pathogens at a time), a wide range of pathogens that can be detected, good specificity and sensitivity, and can directly detect samples without extracting sample nucleic acid;

[0050] 2) The method for rapid detection of bone and joint infection pathogens uses primers and probes specially designed for 9 types of bone and joint infection pathogens. It can achieve high-throughput rapid diagnosis of bone and joint infection pathogens with good specificity and sensitivity, and is easy and quick to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 It is an amplification curve of a standard sample of Staphylococcus aureus of the primers and probes and the detection method for rapid detection of pathogens of bone and joint infection of the present invention;

[0052] Figure 2 It is an amplification curve of the primers and probes for rapid detection of bone and joint infection pathogens and the detection method of the present invention for a standard sample of Acinetobacter baumannii;

[0053] Figure 3 It is an amplification curve of a standard sample of Staphylococcus epidermidis of the primers and probes and the detection method for rapid detection of pathogens of bone and joint infection of the present invention;

[0054] Figure 4 It is an amplification curve of a standard sample of hemolytic Staphylococcus aureus of the primers and probes and the detection method for rapid detection of pathogens of bone and joint infection of the present invention;

[0055] Figure 5 It is an amplification curve of a standard sample of Pseudomonas aeruginosa of the primers and probes and the detection method for rapid detection of pathogens of bone and joint infection of the present invention;

[0056] Figure 6 It is an amplification curve (HEX fluorescence) of the primers and probes for rapid detection of pathogens of bone and joint infection and the detection method of the present invention for a standard sample of Enterobacter cloacae;

[0057] Figure 7 It is an amplification curve (HEX fluorescence) of the primers and probes for rapid detection of bone and joint infection pathogens and the detection method of the present invention for a standard sample of Escherichia coli;

[0058] Figure 8It is an amplification curve of a standard sample of methicillin-resistant Staphylococcus aureus (USA300) of the primers and probes and the detection method for rapid detection of pathogens of bone and joint infection of the present invention;

[0059] Fig. 9 It is an amplification curve of a standard sample of Streptococcus pyogenes of the primers and probes and the detection method for rapid detection of pathogens of bone and joint infection of the present invention;

[0060] Fig.10 It is a threshold detection amplification curve of the primers and probes for rapid detection of bone and joint infection pathogens and the detection method of the present invention for a standard sample of hemolytic Staphylococcus aureus;

[0061] Fig.11 It is a threshold detection amplification curve of the primers and probes for rapid detection of bone and joint infection pathogens and the detection method of the present invention for a standard sample of Staphylococcus aureus SA;

[0062] Fig.12 It is a threshold detection amplification curve of the primers and probes for rapid detection of bone and joint infection pathogens and the detection method of the present invention for a standard sample of Pseudomonas aeruginosa;

[0063] Fig.13 It is a threshold detection amplification curve of the primers and probes for rapid detection of bone and joint infection pathogens and the detection method of the present invention for a standard sample of Acinetobacter baumannii;

[0064] Fig.14 It is a threshold detection amplification curve of the primers and probes for rapid detection of bone and joint infection pathogens and the detection method of the present invention for a standard sample of Staphylococcus epidermidis;

[0065] Fig.15 It is a threshold detection amplification curve of a standard sample of Streptococcus pyogenes of the primers and probes and the detection method for rapid detection of pathogens of bone and joint infection of the present invention;

[0066] Fig.16 It is a threshold detection amplification curve of the primers and probes for rapid detection of bone and joint infection pathogens and the detection method of the present invention for the methicillin-resistant Staphylococcus aureus USA300 standard sample;

[0067] Fig.17 It is a threshold detection amplification curve of the primers and probes for rapid detection of bone and joint infection pathogens and the detection method of the present invention for a standard sample of Enterobacter cloacae;

[0068] Fig.18 It is a threshold detection amplification curve of the primers and probes for rapid detection of bone and joint infection pathogens and the detection method of the present invention for a standard sample of Escherichia coli;

[0069] Fig.19 It is an amplification curve of a sample of a specific application case of the primers and probes for rapid detection of bone and joint infection pathogens and the detection method of the present invention. DETAILED DESCRIPTION

[0070] The technical solution of the present invention is described in detail below in conjunction with the embodiments of the specification.

[0071] The primers and probes for rapid detection of pathogens of bone and joint infection of the present invention include at least three groups of primers and probes for Staphylococcus aureus, Acinetobacter baumannii, Staphylococcus epidermidis, Staphylococcus haemolyticus, Pseudomonas aeruginosa, Enterobacter cloacae, Escherichia coli, methicillin-resistant Staphylococcus aureus and Streptococcus pyogenes;

[0072] The sequence of the upstream primer aureusF for Staphylococcus aureus is shown in SEQ ID No: 1, the sequence of the downstream primer aureusR is shown in SEQ ID No: 2, and the sequence of the probe aureusP is shown in SEQ ID No: 3;

[0073] The sequence of the upstream primer baumanniiF for Acinetobacter baumannii is shown in SEQ ID No: 4, the sequence of the downstream primer baumanniiR is shown in SEQ ID No: 5, and the sequence of the probe baumanniiP is shown in SEQ ID No: 6;

[0074] The sequence of the upstream primer epidermidisF for Staphylococcus epidermidis is shown in SEQ ID No: 7, the sequence of the downstream primer epidermidisR is shown in SEQ ID No: 8, and the sequence of the probe epidermidisP is shown in SEQ ID No: 9;

[0075] The sequence of the upstream primer haemolyticusF for hemolytic Staphylococcus is shown in SEQ ID No: 10, the sequence of the downstream primer haemolyticusR is shown in SEQ ID No: 11, and the sequence of the probe haemolyticusP is shown in SEQ ID No: 12;

[0076] The sequence of the upstream primer aeruginosaF for Pseudomonas aeruginosa is shown in SEQ ID No: 13, the sequence of the downstream primer aeruginosaR is shown in SEQ ID No: 14, and the sequence of the probe aeruginosaP is shown in SEQ ID No: 15;

[0077] The sequence of the upstream primer cloacaeF for Enterobacter cloacae is shown in SEQ ID No: 16, the sequence of the downstream primer cloacaeR is shown in SEQ ID No: 17, and the sequence of the probe cloacaeP is shown in SEQ ID No: 18;

[0078] The sequence of the upstream primer coliF for Escherichia coli is shown in SEQ ID No: 19, the sequence of the downstream primer coliR is shown in SEQ ID No: 20, and the sequence of the probe coliP is shown in SEQ ID No: 21;

[0079] The sequence of the upstream primer mecA-F for methicillin-resistant Staphylococcus aureus is shown in SEQ ID No: 22, the sequence of the downstream primer mecA-R is shown in SEQ ID No: 23, and the sequence of the probe mecA-probe is shown in SEQ ID No: 24;

[0080] The sequence of the upstream primer pyogenesF for Streptococcus pyogenes is shown in SEQ ID No:25, the sequence of the downstream primer pyogenesR is shown in SEQ ID No:26, and the sequence of the probe pyogenesP is shown in SEQ ID No:27.

[0081] A method for rapid detection of pathogens of bone and joint infection mainly comprises the following steps in sequence:

[0082] (i) performing a one-step multiplex RT-qPCR amplification reaction on the sample DNA using at least three sets of primers and probes for Staphylococcus aureus, Acinetobacter baumannii, Staphylococcus epidermidis, Staphylococcus haemolyticus, Pseudomonas aeruginosa, Enterobacter cloacae, Escherichia coli, methicillin-resistant Staphylococcus aureus, and Streptococcus pyogenes;

[0083] The amplification reaction system is as follows:

[0084]

[0085] The final molar concentration of each upstream primer, each downstream primer and each probe is 10 mM, and the sample is tissue fluid of bone tissue, tissue fluid of joint cavity or prosthesis ultrasonic lysate;

[0086] The amplification reaction procedure is as follows:

[0087]

[0088]

[0089] (ii) Result analysis: Whether the patient is infected with the corresponding bone and joint pathogens is determined by detecting whether the PCR reaction product is amplified.

[0090] Product Information:

[0091] Mix: Merlin (including 10× buffer, MgCl2 (25mM), dNTP (25mM), Taq enzyme 5U / μL, 2x lyophilized protective agent, the volume ratio is 5:4:0.4:0.5:15)

[0092] Experimental instrument: Roche real-time fluorescence PCR instrument LightCycler 96

[0093] Standard sample strains: Commercially available

[0094] Experimental data:

[0095] 1. Verification of 9 sets of primers and probes in this application:

[0096] Experimental materials: standard sample strains of 9 bone and joint infection pathogens, primers and probes developed by the present application for 9 bone and joint infection pathogens (Staphylococcus aureus, Acinetobacter baumannii, Staphylococcus epidermidis, Staphylococcus hemolyticus, Pseudomonas aeruginosa, Enterobacter cloacae, Escherichia coli, methicillin-resistant Staphylococcus aureus, and Streptococcus pyogenes), upstream and downstream primers and probes, ddH2O, Mix, fluorescent PCR tubes;

[0097] Detection method: Detection is performed according to the method for rapid detection of bone and joint infection pathogens of the present application.

[0098] The real-time fluorescence PCR (QPCR) results of each sample are shown in Table 3 below:

[0099] Table 3 Real-time fluorescence PCR (QPCR) results of each standard sample strain

[0100]

[0101]

[0102] Depend on Figure 1-9 It can be seen that the 9 sets of primers and probes of the present application can specifically amplify the 9 main bone and joint infection pathogens, including Staphylococcus aureus, Acinetobacter baumannii, Staphylococcus epidermidis, Staphylococcus hemolyticus, Pseudomonas aeruginosa, Enterobacter cloacae, Escherichia coli, methicillin-resistant Staphylococcus aureus and Streptococcus pyogenes, respectively, with good specificity and sensitivity. In the figure, the horizontal axis is the number of amplification cycles, the vertical axis is the Fluorescence amplification multiple, the horizontal line (dashed line) is the baseline for detecting amplification, and when the Fluorescence value is greater than its value, it means that the amplification is successful.

[0103] 2. Detection threshold and specificity detection when 9 sets of primers and probes are mixed in this application

[0104] The 9 sets of primers and probes for 9 bone and joint infection pathogens (Staphylococcus aureus, Acinetobacter baumannii, Staphylococcus epidermidis, Staphylococcus haemolyticus, Pseudomonas aeruginosa, Enterobacter cloacae, Escherichia coli, methicillin-resistant Staphylococcus aureus and Streptococcus pyogenes) were divided into 3 groups, each with three primers and probes, as follows:

[0105] Group 1. Staphylococcus aureus, Acinetobacter baumannii, and methicillin-resistant Staphylococcus aureus (USA300);

[0106] Group 2. Streptococcus pyogenes, Pseudomonas aeruginosa, Escherichia coli;

[0107] Group 3. Staphylococcus haemolyticus, Staphylococcus epidermidis, Enterobacter cloacae.

[0108] The standard sample strains of the 9 bone and joint infection pathogens were added to each group, and the minimum detection threshold of each primer was tested according to the amplification conditions and amplification system of this case. The results are shown in Table 4 and Figure 10-18 (The horizontal axis is the number of amplification cycles, and the vertical axis is the CT value).

[0109] From Table 4 and Figure 10-18 It can be seen that the nine sets of primers and probes have good amplification specificity for each bacterium, and no other expansion curves appear. The minimum detection threshold of each bacterium is 10 3 CFU. Fig.16 The reason for the two amplification curves is that MRSA contains some genes of common S. aureus.

[0110] It can be seen that the 9 sets of primers and probes in this case can specifically amplify 9 bone and joint infection pathogens (Staphylococcus aureus, Acinetobacter baumannii, Staphylococcus epidermidis, Staphylococcus haemolyticus, Pseudomonas aeruginosa, Enterobacter cloacae, Escherichia coli, methicillin-resistant Staphylococcus aureus and Streptococcus pyogenes) when used in combination, and the detection sensitivity is good.

[0111] Table 4 Specific mixed application of the amplification effect of each standard strain test results

[0112]

[0113] III. Clinical application test of the 9 sets of primers and probes in this application

[0114] 1. During the operation, the synovial tissue, joint fluid or ultrasonic lysate obtained after ultrasonic lysis of the patient's periprosthetic infection was taken as samples for testing. A total of 65 patients were sampled. First, each sample was placed in a sterile container and lysozyme was added to dissolve the bacterial cell wall, then added to the assembled reaction container, and finally added to the 9 sets of primers and probes in this case. The amplification system and procedure of this case were used for amplification detection. The standards formulated by the Microbiology and Infection Society (MSIS) in 2018 were used as the "gold standard" for the diagnosis of prosthetic joint infection (PJI). The sensitivity and specificity of microbial culture and POCT detection in this case in the diagnosis of PJI were compared and analyzed. The results are shown in Table 5:

[0115] Table 5 Statistics of sample test results

[0116]

[0117] Sensitivity, also known as True Positive Rate (TPR), refers to the proportion of samples that are correctly identified as positive among samples that are actually positive. It measures the ability of the test to identify true cases, that is, the probability that the patient is judged to be positive. The calculation formula for sensitivity is: Sensitivity = True Positive (TP) / (True Positive (TP) + False Negative (FN)).

[0118] Specificity, also known as the True Negative Rate (TNR), refers to the proportion of samples that are correctly identified as negative among samples that are actually negative. It measures the ability of the test to correctly exclude individuals without the disease, that is, the proportion of negative test results. The calculation formula for specificity is: Specificity = True Negative (TN) / (True Negative (TN) + False Positive (FP)).

[0119] The two detection methods were used to detect patients in the periprosthetic infection group and the aseptic infection group. The POCT method detected 31 positive patients (with bacteria) in the periprosthetic infection group and 2 positive patients in the aseptic loosening group. The microbial culture method detected 34 positive patients in the periprosthetic infection group. The sensitivity of the sample microbial culture was 85.00%, while the sensitivity of the POCT detection in this case was 77.50%. The t-test was used to compare the sensitivity of the two groups, and P>0.05 showed no difference in sensitivity between the two groups. In terms of specificity, the specificity of microbial culture reached 100.00%, while the specificity of the POCT detection in this case was 90.00%. The t-test was used to compare the sensitivity of the two groups, and P>0.05 showed no difference in specificity between the two groups. Overall, there was no significant difference in diagnostic efficacy between the two. It can be seen that the feasibility of POCT detection in this case.

[0120] 2. Test the joint fluid of one of the patients:

[0121] The 9 sets of primers and probes for 9 bone and joint infection pathogens (Staphylococcus aureus, Acinetobacter baumannii, Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, Enterobacter cloacae, Escherichia coli, methicillin-resistant Staphylococcus aureus, and Streptococcus pyogenes) in this case were divided into three groups, namely: Group 1. Staphylococcus aureus, Staphylococcus aureus, Streptococcus pyogenes; Group 2. Acinetobacter baumannii, Staphylococcus epidermidis, Pseudomonas aeruginosa; Group 3. Methicillin-resistant Staphylococcus aureus (USA 300), Enterobacter cloacae, and Escherichia coli. The samples were added to each group and tested according to the amplification conditions and amplification system of this case. The test results are as follows. Fig.19 (The horizontal axis is the number of amplification cycles, and the vertical axis is the CT value). The upper curve is the amplification curve of Staphylococcus aureus, and the lower curve is the amplification curve of Staphylococcus aureus. It can be seen that the patient was infected with Acinetobacter baumannii and Staphylococcus aureus. The test results are consistent with the results of bacterial culture.

[0122] The primers and probes for rapid detection of pathogens of bone and joint infection and the detection method described in the present invention are not limited to the above-mentioned embodiments. Any improvement or replacement based on the principle of the present invention should be within the protection scope of the present invention.

Claims

1. A primer and probe for rapid detection of pathogens of bone and joint infection, characterized in that: The method comprises at least three sets of primers and probes for Staphylococcus aureus, Acinetobacter baumannii, Staphylococcus epidermidis, Staphylococcus haemolyticus, Pseudomonas aeruginosa, Enterobacter cloacae, Escherichia coli, methicillin-resistant Staphylococcus aureus and Streptococcus pyogenes; The sequence of the upstream primer aureusF for Staphylococcus aureus is shown in SEQ ID No: 1, the sequence of the downstream primer aureusR is shown in SEQ ID No: 2, and the sequence of the probe aureusP is shown in SEQ ID No: 3; The sequence of the upstream primer baumanniiF for Acinetobacter baumannii is shown in SEQ ID No: 4, the sequence of the downstream primer baumanniiR is shown in SEQ ID No: 5, and the sequence of the probe baumanniiP is shown in SEQ ID No: 6; The sequence of the upstream primer epidermidisF for Staphylococcus epidermidis is shown in SEQ ID No: 7, the sequence of the downstream primer epidermidisR is shown in SEQ ID No: 8, and the sequence of the probe epidermidisP is shown in SEQ ID No: 9; The sequence of the upstream primer haemolyticusF for hemolytic Staphylococcus is shown in SEQ ID No: 10, the sequence of the downstream primer haemolyticusR is shown in SEQ ID No: 11, and the sequence of the probe haemolyticusP is shown in SEQ ID No: 12; The sequence of the upstream primer aeruginosaF for Pseudomonas aeruginosa is shown in SEQ ID No: 13, the sequence of the downstream primer aeruginosaR is shown in SEQ ID No: 14, and the sequence of the probe aeruginosaP is shown in SEQ ID No: 15; The sequence of the upstream primer cloacaeF for Enterobacter cloacae is shown in SEQ ID No: 16, the sequence of the downstream primer cloacaeR is shown in SEQ ID No: 17, and the sequence of the probe cloacaeP is shown in SEQ ID No: 18; The sequence of the upstream primer coliF for Escherichia coli is shown in SEQ ID No: 19, the sequence of the downstream primer coliR is shown in SEQ ID No: 20, and the sequence of the probe coliP is shown in SEQ ID No: 21; The sequence of the upstream primer mecA-F for methicillin-resistant Staphylococcus aureus is shown in SEQ ID No: 22, the sequence of the downstream primer mecA-R is shown in SEQ ID No: 23, and the sequence of the probe mecA-probe is shown in SEQ ID No: 24; The sequence of the upstream primer pyogenesF for Streptococcus pyogenes is shown in SEQ ID No:25, the sequence of the downstream primer pyogenesR is shown in SEQ ID No:26, and the sequence of the probe pyogenesP is shown in SEQ ID No:

27.

2. A method for rapid detection of pathogens of bone and joint infection, characterized in that: It mainly includes the following steps in order: (i) performing a one-step multiplex RT-qPCR amplification reaction on the sample DNA using at least three sets of primers and probes for Staphylococcus aureus, Acinetobacter baumannii, Staphylococcus epidermidis, Staphylococcus haemolyticus, Pseudomonas aeruginosa, Enterobacter cloacae, Escherichia coli, methicillin-resistant Staphylococcus aureus, and Streptococcus pyogenes as described in claim 1; The amplification reaction system is as follows: The final molar concentration of each upstream primer, each downstream primer and each probe is 10 mM, and the sample is tissue fluid of bone tissue, tissue fluid of joint cavity or prosthesis ultrasonic lysate; The amplification reaction procedure is as follows: (ii) Result analysis: Whether the patient is infected with the corresponding bone and joint pathogens is determined by detecting whether the PCR reaction product is amplified.

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