Primer probe composition, kit and method for detecting common pathogens in orthopedics department

Abstract

The invention discloses a primer probe composition, a kit and a method for detecting common pathogens in the orthopedics department, and relates to the technical field of molecular biology diagnosis. A plurality of bacteria and fungi (including staphylococcus aureus, streptococcus, methicillin-resistant staphylococcus aureus, escherichia coli, pseudomonas, candida and aspergillus) commonly seen in orthopedic infection can be detected at the same time, and the detection coverage range and efficiency are remarkably improved; moreover, high sensitivity and specificity are ensured through optimized reaction conditions and enhanced signal detection, and accurate diagnostic information can be quickly provided for doctors.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biological diagnosis, and in particular to a primer-probe combination, a kit and a method for detecting common orthopedic pathogens. Background Art

[0002] In modern medical practice, rapid and accurate diagnosis of orthopedic-related pathogen infections is crucial to treatment effectiveness and patient recovery. Orthopedic infections include but are not limited to osteomyelitis, arthritis, etc., often involving a variety of bacteria, fungi and other microorganisms. Their diagnosis is extremely challenging and requires highly accurate and rapid detection methods.

[0003] At present, the diagnosis of orthopedic pathogens mainly relies on microbial culture and molecular biology methods. Microbial culture is the traditional gold standard, but it is time-consuming and has insufficient sensitivity for certain difficult-to-culture microorganisms. Molecular biological methods such as polymerase chain reaction (PCR) provide faster detection speeds, but existing PCR kits are usually limited in specificity and cannot cover the many pathogens commonly found in orthopedic infections. Existing detection methods cannot meet the clinical needs for rapid, comprehensive and accurate diagnosis of orthopedic pathogens. This leads to delayed diagnosis, affecting treatment decisions and patient prognosis. Therefore, there is an urgent need for a new method that can quickly and accurately detect a variety of orthopedic pathogens.

[0004] Based on these problems, the development of a PCR kit that can quickly and accurately detect multiple orthopedic pathogens has become a research focus. This new kit needs to have high sensitivity and specificity and be able to provide reliable diagnostic results within hours to guide clinical treatment. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a primer-probe combination, a kit and a method for detecting common orthopedic pathogens, which can simultaneously detect multiple bacteria and fungi commonly found in orthopedic infections, significantly improving the coverage and efficiency of detection, and has high sensitivity and good specificity, and can provide doctors with accurate diagnostic information in a short time, so that doctors can quickly make more effective treatment decisions.

[0006] The technical problem to be solved by the present invention is achieved by adopting the following technical solutions:

[0007] The first object of the present invention is to provide a primer-probe combination for detecting Staphylococcus aureus, Streptococcus, methicillin-resistant Staphylococcus aureus, Escherichia coli, Pseudomonas, Candida and Aspergillus.

[0008] The second object of the present invention is to provide a kit for detecting Staphylococcus aureus, Streptococcus, methicillin-resistant Staphylococcus aureus, Escherichia coli, Pseudomonas, Candida and Aspergillus, comprising the primer-probe combination.

[0009] The third object of the present invention is to provide the use of the kit in the detection of common pathogens in orthopedics.

[0010] The fourth object of the present invention is to provide a method for detecting common orthopedic pathogens, using the DNA of the pathogen in the sample to be tested as a template, using the kit to perform a fluorescent quantitative PCR amplification reaction, and collecting fluorescent signals to determine the content of Staphylococcus aureus, Streptococcus, methicillin-resistant Staphylococcus aureus, Escherichia coli, Pseudomonas, Candida, and Aspergillus in the sample to be tested.

[0011] The beneficial effects of the present invention are as follows: the kit of the present invention adopts a unique primer and probe design, which can simultaneously detect a variety of bacteria and fungi commonly found in orthopedic infections, significantly improving the coverage and efficiency of detection, and ensuring high sensitivity and specificity through optimized reaction conditions and enhanced signal detection, and can quickly provide doctors with accurate diagnostic information. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Schematic diagram of the sensitivity amplification of Staphylococcus aureus in the FAM channel (the concentration of nucleic acid standards from left to right is 2×10 6 Copy number / μL, 2×10 5 Copy number / μL, 2×10 4 Copy number / μL, 2×10 3 Copy number / μL, 2×10 2 Copy number / μL, 2×10 1 Copy number / μL, 2×10 0 copies / μL);

[0013] Figure 2 Schematic diagram of the sensitivity amplification of Streptococcus in the ROX channel (the concentration of nucleic acid standards from left to right is 2×10 6 Copy number / μL, 2×10 5 Copy number / μL, 2×10 4 Copy number / μL, 2×10 3 Copy number / μL, 2×10 2 Copy number / μL, 2×10 1 Copy number / μL, 2×10 0 copies / μL);

[0014] Figure 3Schematic diagram of sensitivity amplification of methicillin-resistant Staphylococcus aureus in the VIC channel (the concentration of nucleic acid standards from left to right is 2×10 6 Copy number / μL, 2×10 5 Copy number / μL, 2×10 4 Copy number / μL, 2×10 3 Copy number / μL, 2×10 2 Copy number / μL, 2×10 1 Copy number / μL, 2×10 0 copies / μL);

[0015] Figure 4 Schematic diagram of sensitivity amplification of E. coli in the TAMRA channel (the concentration of nucleic acid standards from left to right is 2×10 6 Copy number / μL, 2×10 5 Copy number / μL, 2×10 4 Copy number / μL, 2×10 3 Copy number / μL, 2×10 2 Copy number / μL, 2×10 1 Copy number / μL, 2×10 0 copies / μL);

[0016] Figure 5 Schematic diagram of sensitivity amplification of Pseudomonas in the VIC channel (the concentration of nucleic acid standards from left to right is 2×10 6 Copy number / μL, 2×10 5 Copy number / μL, 2×10 4 Copy number / μL, 2×10 3 Copy number / μL, 2×10 2 Copy number / μL, 2×10 1 Copy number / μL, 2×10 0 copies / μL);

[0017] Figure 6 Schematic diagram of sensitivity amplification of Candida species in the FAM channel (the concentration of nucleic acid standards from left to right is 2×10 6 Copy number / μL, 2×10 5 Copy number / μL, 2×10 4 Copy number / μL, 2×10 3 Copy number / μL, 2×10 2 Copy number / μL, 2×10 1 Copy number / μL, 2×10 0 copies / μL);

[0018] Figure 7Schematic diagram of sensitivity amplification of Aspergillus in the ROX channel (the concentration of nucleic acid standards from left to right is 2×10 5 Copy number / μL, 2×10 4 Copy number / μL, 2×10 3 Copy number / μL, 2×10 2 Copy number / μL, 2×10 1 Copy number / μL, 2×10 0 copies / μL). DETAILED DESCRIPTION

[0019] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further described below in conjunction with specific embodiments and diagrams.

[0020] The present invention provides a primer-probe composition for detecting Staphylococcus aureus, Streptococcus, methicillin-resistant Staphylococcus aureus, Escherichia coli, Pseudomonas, Candida and Aspergillus, comprising the following primers and probes:

[0021] The upstream primer sequence of Staphylococcus aureus is shown in SEQ ID NO.1, the downstream primer sequence is shown in SEQ ID NO.2, and the probe sequence is shown in SEQ ID NO.3;

[0022] The upstream primer sequence of the Streptococcus is shown in SEQ ID NO.4, the downstream primer sequence is shown in SEQ ID NO.5, and the probe sequence is shown in SEQ ID NO.6;

[0023] The upstream primer sequence of the methicillin-resistant Staphylococcus aureus is shown in SEQ ID NO.7, the downstream primer sequence is shown in SEQ ID NO.8, and the probe sequence is shown in SEQ ID NO.9;

[0024] The upstream primer sequence of the Escherichia coli is shown in SEQ ID NO.10, the downstream primer sequence is shown in SEQ ID NO.11, and the probe sequence is shown in SEQ ID NO.12;

[0025] The upstream primer sequence of the Pseudomonas is shown in SEQ ID NO.13, the downstream primer sequence is shown in SEQ ID NO.14, and the probe sequence is shown in SEQ ID NO.15;

[0026] The upstream primer sequence of the Candida species is shown in SEQ ID NO.16, the downstream primer sequence is shown in SEQ ID NO.17, and the probe sequence is shown in SEQ ID NO.18;

[0027] The upstream primer sequence of the Aspergillus genus is shown in SEQ ID NO.19, the downstream primer sequence is shown in SEQ ID NO.20, and the probe sequence is shown in SEQ ID NO.21.

[0028] Furthermore, one end of the probe sequence is labeled with a fluorescent group, and the other end is labeled with a quencher group. The 5' end of the probe sequence may be labeled with a fluorescent group, and the 3' end may be labeled with a quencher group; or the 3' end of the probe sequence may be labeled with a fluorescent group, and the 5' end may be labeled with a quencher group.

[0029] Furthermore, the fluorescent group is selected from one of FAM, VIC, TARAM, ROX, and CY5; and the quenching group is selected from one of BHQ1, BHQ2, and BHQ3.

[0030] In a specific embodiment, the 5′ end of the Staphylococcus aureus probe is labeled with a fluorescent group FAM, and the 3′ end is labeled with a quencher group BHQ1; the 5′ end of the Streptococcus probe is labeled with a fluorescent group ROX, and the 3′ end is labeled with a quencher group BHQ2; the 5′ end of the methicillin-resistant Staphylococcus aureus probe is labeled with a fluorescent group VIC, and the 3′ end is labeled with a quencher group BHQ3; the 5′ end of the Escherichia coli probe is labeled with a fluorescent group TAMRA, and the 3′ end is labeled with a quencher group BHQ1; the 5′ end of the Pseudomonas probe is labeled with a fluorescent group VIC, and the 3′ end is labeled with a quencher group BHQ1; the 5′ end of the Candida probe is labeled with a fluorescent group FAM, and the 3′ end is labeled with a quencher group BHQ2; the 5′ end of the Aspergillus probe is labeled with a fluorescent group ROX, and the 3′ end is labeled with a quencher group BHQ3.

[0031] Table 1

[0032]

[0033]

[0034] The invention provides a kit for detecting Staphylococcus aureus, Streptococcus, methicillin-resistant Staphylococcus aureus, Escherichia coli, Pseudomonas, Candida and Aspergillus, comprising the primer-probe combination.

[0035] Furthermore, the primer-probe combination is dissolved in enzyme-free sterile water, the concentration of the upstream primer and the downstream primer is 0.2-0.5 μM, and the concentration of the probe is 100-200 nM.

[0036] Furthermore, the kit also includes independently packaged Staphylococcus aureus nucleic acid standards, Streptococcus nucleic acid standards, methicillin-resistant Staphylococcus aureus nucleic acid standards, Escherichia coli nucleic acid standards, Pseudomonas nucleic acid standards, Candida nucleic acid standards and Aspergillus nucleic acid standards, the sequence of the Staphylococcus aureus nucleic acid standard is shown in SEQ ID NO.22; the sequence of the Streptococcus nucleic acid standard is shown in SEQ ID NO.23; the sequence of the methicillin-resistant Staphylococcus aureus nucleic acid standard is shown in SEQ ID NO.24; the sequence of the Escherichia coli nucleic acid standard is shown in SEQ ID NO.25; the sequence of the Pseudomonas nucleic acid standard is shown in SEQ ID NO.26; the sequence of the Candida nucleic acid standard is shown in SEQ ID NO.27; the sequence of the Aspergillus nucleic acid standard is shown in SEQ ID NO.28.

[0037] Table 2

[0038]

[0039]

[0040]

[0041] Furthermore, the kit also includes a PCR reaction solution and TaqDNA polymerase.

[0042] The present invention provides application of the kit in detecting common pathogens in orthopedics.

[0043] The present invention provides a method for detecting common orthopedic pathogens, using the DNA of the pathogen in the sample to be tested as a template, using the kit to perform a fluorescent quantitative PCR amplification reaction, and collecting fluorescent signals to determine the content of Staphylococcus aureus, Streptococcus, methicillin-resistant Staphylococcus aureus, Escherichia coli, Pseudomonas, Candida, and Aspergillus in the sample to be tested. Specifically, the following steps are included:

[0044] (1) extracting nucleic acid from the sample to be tested;

[0045] (2) adding the nucleic acid extracted from the sample to the kit for fluorescence quantitative PCR amplification reaction and collecting the fluorescence signal;

[0046] (3) Determine whether the sample contains Staphylococcus aureus, Streptococcus, methicillin-resistant Staphylococcus aureus, Escherichia coli, Pseudomonas, Candida, or Aspergillus based on the fluorescence signal.

[0047] Dispense the primers and probes for each of the above pathogens into designated amplification tubes corresponding to fixed positions. Therefore, it is only necessary to detect the corresponding fluorescence in the corresponding amplification tubes. For example, Staphylococcus aureus and Candida spp. that both use the FAM fluorescence channel are located in different amplification tubes and do not affect each other. The determination method after the fluorescence quantitative PCR amplification reaction is as follows: If the Ct value of the test sample in the FAM channel is ≤ 35 and there is an amplification curve, it is judged as positive; if the Ct value in the ROX channel is ≤ 35 and there is an amplification curve, it is judged as positive; if the Ct value in the VIC channel is ≤ 35 and there is an amplification curve, it is judged as positive; if the Ct value in the TAMRA channel is ≤ 35 and there is an amplification curve, it is judged as positive. If no Ct value is detected or Ct > 38 in all channels, it is judged as negative. If the Ct value of the test sample in any channel is 35 < Ct ≤ 38, the nucleic acid of the sample should be re-extracted and tested again. For the repeated test results, if there is no Ct value, it is negative; otherwise, it is positive.

[0048] In a specific embodiment, dilute the nucleic acid standards of Staphylococcus aureus, Streptococcus spp., methicillin-resistant Staphylococcus aureus, Escherichia coli, Pseudomonas spp., and Candida spp. to 2×10 0 copies / μL, 2×10 1 copies / μL, 2×10 2 copies / μL, 2×10 3 copies / μL, 2×10 4 copies / μL, 2×10 5 copies / μL, 2×10 6 copies / μL; dilute the nucleic acid standard of Aspergillus spp. to 2×10 0 copies / μL, 2×10 1 copies / μL, 2×10 2 copies / μL, 2×10 3 copies / μL, 2×10 4 copies / μL, 2×10 5 copies / μL to prepare templates with different concentrations. The positive control is a standard premix of 2×10 6 copies / μL or 2×10 5 copies / μL, and the negative control is enzyme-free and sterile water (RNase / DNase free water).

[0049] Configure the qPCR reaction system:

[0050] Table 3

[0051] Reagents Reaction dosage 2×qPCR reaction solution 10μL TaqDNA polymerase (5U / μL) 0.4μL Upstream primer 0.5 μM, downstream primer 0.5 μM, probe 200 nM 2μL Template (nucleic acid extracted from a sample) 2μL Enzyme-free sterile water Make up to 20 μL

[0052] The qPCR reaction solution was the qPCR premix from Thermo Fisher Scientific (PowerUp TM SYBR TM Green Master Mix) and TaqDNA polymerase was Qiagen's TaqDNA polymerase (HotStarTaq DNA Polymerase).

[0053] The fluorescence quantitative PCR amplification reaction procedure is as follows:

[0054] Step 1: 95°C, 2-3 min;

[0055] Step 2: 95°C, 15-30 seconds;

[0056] Step 3: 62°C, 30 seconds;

[0057] Step 4: 72°C, 30 seconds;

[0058] Repeat steps 2-4 for a total of 40 cycles;

[0059] Step 5: 72°C, 5-10 min;

[0060] Step 6: Store at 4°C.

[0061] The fluorescence signal was detected during the annealing extension at 62°C to obtain the amplification curve of each concentration standard. The PCR reaction was repeated 3 times for each concentration standard.

[0062] According to the amplification curve ( Figures 1 to 7 ), a standard curve was prepared with the Cq value obtained for each standard concentration as the ordinate and the logarithm of the standard copy number as the abscissa, where Cq was the number of cycles required for the fluorescence signal in each amplification tube to reach the set threshold, and the threshold was automatically generated by the instrument. The corresponding standard curves are statistically shown in Table 4.

[0063] Table 4

[0064]

[0065] The nucleic acid standards of the above 7 pathogens were 2×10 0 ~2×10 6 Copies / μL or 2×10 0 ~2×10 5 The linear relationship is very good within the copy number / μL concentration range, and the sensitivity is high. Figures 1 to 7 As shown, the detection specificity of the above 7 pathogens was good, and no false positives occurred.

[0066] The ST cell culture medium contaminated with Staphylococcus aureus, Streptococcus, methicillin-resistant Staphylococcus aureus, Escherichia coli, Pseudomonas, Candida, and Aspergillus was used for verification. The specific steps are as follows:

[0067] Take 500μL of ST cell culture medium after centrifugation and concentration into a 1.5mL centrifuge tube, add Staphylococcus aureus standard, Streptococcus standard, methicillin-resistant Staphylococcus aureus standard, Escherichia coli standard, Pseudomonas standard, Candida standard, Aspergillus standard, then add 500μL TRIZOL reagent and shake well, let stand at room temperature for 10 minutes, add 200μL chloroform after standing, shake well after shaking for 30s. Centrifuge at 4℃ and 12000rpm for 15min, remove the supernatant and transfer to a clean centrifuge tube. Add an equal volume of isopropanol, then mix the liquid and let stand at room temperature for 15min. Centrifuge at 4℃ and 12000rpm for 15min, discard the supernatant, and add pre-cooled 75% ethanol solution to the precipitate. Centrifuge at 4°C and 12,000 rpm for 15 min, remove all supernatant with a pipette, and place the precipitate in a biosafety cabinet to dry at room temperature for 15 min. Finally, add 20 μL DEPC to dissolve the precipitate and store at -20°C.

[0068] The fluorescence quantitative PCR amplification reaction was performed according to the above reaction system and reaction procedure, and the clean culture medium was used as the negative control to obtain the amplification curve of each pathogen in the sample to be tested. The negative control had no amplification curve.

[0069] Substitute the Cq values ​​of the amplification curves of each pathogen in the sample to be tested into the standard curve, and calculate the content of Staphylococcus aureus in the sample to be tested to be 1.41×10 6 The number of copies / μL of Streptococcus was 3.49×10 5 The copy number / μL of methicillin-resistant Staphylococcus aureus was 5.13×10 5 The number of copies / μL of E. coli was 1.38×10 6 The number of copies / μL of Pseudomonas was 4.13×10 5 The copy number / μL of Candida species was 2.14×10 5 The copy number / μL of Aspergillus spp. was 1.36×10 5 Copy number / μL.

[0070] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A primer-probe combination for detecting Staphylococcus aureus, Streptococcus, methicillin-resistant Staphylococcus aureus, Escherichia coli, Pseudomonas, Candida and Aspergillus, It is characterized in that The following primers and probes are included: The upstream primer sequence of Staphylococcus aureus is shown in SEQ ID NO.1, the downstream primer sequence is shown in SEQ ID NO.2, and the probe sequence is shown in SEQ ID NO.3; The upstream primer sequence of the Streptococcus is shown in SEQ ID NO.4, the downstream primer sequence is shown in SEQ ID NO.5, and the probe sequence is shown in SEQ ID NO.6; The upstream primer sequence of the methicillin-resistant Staphylococcus aureus is shown in SEQ ID NO.7, the downstream primer sequence is shown in SEQ ID NO.8, and the probe sequence is shown in SEQ ID NO.9; The upstream primer sequence of the Escherichia coli is shown in SEQ ID NO.10, the downstream primer sequence is shown in SEQ ID NO.11, and the probe sequence is shown in SEQ ID NO.12; The upstream primer sequence of the Pseudomonas is shown in SEQ ID NO.13, the downstream primer sequence is shown in SEQ ID NO.14, and the probe sequence is shown in SEQ ID NO.15; The upstream primer sequence of the Candida species is shown in SEQ ID NO.16, the downstream primer sequence is shown in SEQ ID NO.17, and the probe sequence is shown in SEQ ID NO.18; The upstream primer sequence of the Aspergillus genus is shown in SEQ ID NO.19, the downstream primer sequence is shown in SEQ ID NO.20, and the probe sequence is shown in SEQ ID NO.

21.

2. The primer-probe combination according to claim 1, Features: One end of the probe sequence is labeled with a fluorescent group, and the other end is labeled with a quencher group.

3. The primer-probe composition according to claim 2, Features: The fluorescent group is selected from one of FAM, VIC, TARAM, ROX, and CY5; the quenching group is selected from one of BHQ1, BHQ2, and BHQ3.

4. The primer-probe combination according to claim 3, Features: The 5′ end of the Staphylococcus aureus probe is labeled with a fluorescent group FAM, and the 3′ end is labeled with a quencher group BHQ1; the 5′ end of the Streptococcus probe is labeled with a fluorescent group ROX, and the 3′ end is labeled with a quencher group BHQ2; the 5′ end of the methicillin-resistant Staphylococcus aureus probe is labeled with a fluorescent group VIC, and the 3′ end is labeled with a quencher group BHQ3; the 5′ end of the Escherichia coli probe is labeled with a fluorescent group TAMRA, and the 3′ end is labeled with a quencher group BHQ1; the 5′ end of the Pseudomonas probe is labeled with a fluorescent group VIC, and the 3′ end is labeled with a quencher group BHQ1; the 5′ end of the Candida probe is labeled with a fluorescent group FAM, and the 3′ end is labeled with a quencher group BHQ2; the 5′ end of the Aspergillus probe is labeled with a fluorescent group ROX, and the 3′ end is labeled with a quencher group BHQ3.

5. A kit for detecting Staphylococcus aureus, Streptococcus, methicillin-resistant Staphylococcus aureus, Escherichia coli, Pseudomonas, Candida and Aspergillus, comprising the primer-probe combination according to any one of claims 1 to 4.

6. The kit according to claim 5, Features: The kit also includes independently packaged Staphylococcus aureus nucleic acid standards, Streptococcus nucleic acid standards, methicillin-resistant Staphylococcus aureus nucleic acid standards, Escherichia coli nucleic acid standards, Pseudomonas nucleic acid standards, Candida nucleic acid standards and Aspergillus nucleic acid standards; The sequence of the Staphylococcus aureus nucleic acid standard is shown in SEQ ID NO.22; The sequence of the Streptococcus nucleic acid standard is shown in SEQ ID NO.23; The sequence of the methicillin-resistant Staphylococcus aureus nucleic acid standard is shown in SEQ ID NO.24; The sequence of the Escherichia coli nucleic acid standard is shown in SEQ ID NO.25; The sequence of the Pseudomonas nucleic acid standard is shown in SEQ ID NO.26; The sequence of the Candida nucleic acid standard is shown in SEQ ID NO.27; The sequence of the Aspergillus nucleic acid standard is shown in SEQ ID NO.

28.

7. The kit according to claim 6, Features: The kit also includes PCR reaction solution and TaqDNA polymerase.

8. Use of the kit according to any one of claims 5 to 7 in the detection of common pathogens in orthopedics.

9. A method for detecting common pathogens in orthopedics. Features: Using the DNA of the pathogen in the sample to be tested as a template, a fluorescent quantitative PCR amplification reaction is performed using the kit described in any one of claims 5 to 7, and the fluorescent signal is collected to determine the content of Staphylococcus aureus, Streptococcus, methicillin-resistant Staphylococcus aureus, Escherichia coli, Pseudomonas, Candida, and Aspergillus in the sample to be tested.

10. The method according to claim 9, It is characterized in that The fluorescence quantitative PCR amplification reaction procedure is as follows: Step 1: 95°C, 2-3 min; Step 2: 95°C, 15-30 seconds; Step 3: 62°C, 30 seconds; Step 4: 72°C, 30 seconds; Repeat steps 2-4 for a total of 40 cycles; Step 5: 72°C, 5-10 min; Step 6: Store at 4°C.

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