Multiple qPCR (quantitative polymerase chain reaction) detection reagent for detecting spinal infection pathogens

Abstract

The invention discloses a multiplex qPCR (quantitative polymerase chain reaction) detection reagent for detecting spinal infection pathogens. The multiplex qPCR detection reagent comprises specific primers and probes for detecting staphylococcus aureus, staphylococcus epidermidis, escherichia coli, mycobacterium tuberculosis, brucella, klebsiella pneumoniae and streptococcus agalactiae; according to the invention, different fluorescently-labeled probes are utilized to add specific primers and probes of multiple pathogens into a reaction system to detect multiple targets at the same time, and the method has the advantages of good specificity, high sensitivity, short detection period, capability of simultaneously detecting multiple pathogens, rapidness, simplicity, convenience and economy; a convenient method is provided for rapid detection of gynecological pathogens, and the kit has important significance in epidemiological research, clinical early molecular diagnosis and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a multiplex qPCR detection reagent for detecting pathogens causing spinal infections, and uses the multiplex qPCR detection reagent to detect 7 kinds of pathogenic bacteria causing spinal infections. Background Art

[0002] Spinal Infections (SI) are infectious diseases of the spinal region caused by bacteria or other pathogens, usually affecting the vertebrae, intervertebral discs, spinal cord or surrounding tissues. Common types of spinal infections include vertebral osteomyelitis, discitis and epidural abscess. The incidence of spinal infections in developed countries is only 4-24 cases per million people per year, and the proportion is higher in developing countries due to the high incidence of tuberculosis. In recent years, due to the influence of factors such as advanced age, malnutrition, diabetes, intravenous drug abuse, popularization of the use of implants in the cardiovascular and urogenital systems, immunosuppression, HIV, sepsis and long-term use of hormones, its incidence has gradually increased and has become a global health problem.

[0003] Spinal infections can be caused by a single microorganism (monomicrobial infection) or by multiple microorganisms co-infecting (polymicrobial infection). Polymicrobial infections are usually more difficult to control and treat because of complementary cross-resistance between different bacteria. The main route of infection is hematogenous dissemination, followed by iatrogenic or traumatic infections. Among them, Staphylococcus aureus (SA) is the most common pathogen causing spinal infections. This bacterium often causes suppurative infections of the intervertebral disc and vertebral body, usually accompanied by obvious local inflammation and abscess formation. Enterobacteriaceae are also common bacteria causing spinal infections, and about 7 - 33% of suppurative spinal infections are caused by Enterobacteriaceae. Among them, the most common is Escherichia coli (EC). Coagulase Negative Staphylococci (CoNS) are also common pathogenic bacteria in spinal infections, accounting for 5% - 16% of the total number of cases. Among them, Staphylococcus epidermidis is a common bacterium, which may be related to in vivo implants and postoperative infections. Brucellosis caused by Brucella spp. (Br), as an important zoonosis, has a high incidence in endemic areas such as the Mediterranean Basin, Latin America, the Middle East, parts of Africa, and West Asia. Epidemiological studies have shown that spinal infections caused by this pathogen account for about 21% - 48% of all spinal infection cases. Its main route of transmission is the ingestion of unpasteurized contaminated dairy products or direct contact with infected animals. Mycobacterium tuberculosis (MTB), as one of the main pathogenic bacteria causing spinal infections globally, has significant geographical differences in its epidemiological characteristics. Epidemiological survey data show that in developed countries, spinal infections caused by Mycobacterium tuberculosis account for about 9% - 46% of all spinal infection cases.

[0004] Clinically common detection methods include immunological methods, smear microscopy, and isolation and culture methods, which have the disadvantages of low detection rate, poor specificity, and long time consumption. Conventional qPCR only detects a single index in one reaction tube. If there are many detection indexes or many samples to be detected, it is undoubtedly a huge waste of qPCR reagents and samples. In multiplex qPCR, multiple targets are amplified and detected in a single tube; therefore, the expression levels of several targets or target genes can be quickly measured. This method of simultaneously detecting multiple indexes in a single reaction tube greatly reduces the consumption of related reagents. Even if there is enough qPCR enzyme, multiplex qPCR will also save sample processing time and other reagent consumables. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention provides a multiplex qPCR detection reagent for detecting pathogens causing spinal infections, which includes specific primers and probes for detecting Staphylococcus aureus (SA), Staphylococcus epidermidis (SE), Escherichia coli (EC), Mycobacterium tuberculosis (MTB), Streptococcus agalactiae (SA), Brucella (Br), and Klebsiella pneumoniae (KP). The detection reagent of the present invention also includes other conventional reagents for multiplex qPCR. The present invention evaluates the specificity, sensitivity, repeatability, and accuracy of the established method and finds that the multiplex qPCR method has the advantages of high detection sensitivity, strong specificity, good repeatability, low requirements for instrument equipment, simple operation, and short required time. Therefore, it has great application value, provides a convenient method for the rapid detection of pathogens causing spinal infections, and is of great significance for epidemiological research and early clinical molecular diagnosis, etc.

[0006] The specific primers are SEQ ID NO:1 and SEQ ID NO:2 for Staphylococcus aureus, SEQ ID NO:4 and SEQ ID NO:5 for Staphylococcus epidermidis, SEQ ID NO:7 and SEQ ID NO:8 for Escherichia coli, SEQ ID NO:10 and SEQ ID NO:11 for Mycobacterium tuberculosis, SEQ ID NO:13 and SEQ ID NO:14 for Brucella, SEQ ID NO:16 and SEQ ID NO:17 for Klebsiella pneumoniae, and SEQ ID NO:19 and SEQ ID NO:20 for Streptococcus agalactiae;

[0007] The probes are SEQ ID NO:3 for Staphylococcus aureus, SEQ ID NO:6 for Staphylococcus epidermidis, SEQ ID NO:9 for Escherichia coli, SEQ ID NO:12 for Mycobacterium tuberculosis, SEQ ID NO:15 for Brucella, SEQ ID NO:18 for Klebsiella pneumoniae, and SEQ ID NO:21 for Streptococcus agalactiae.

[0008] The above detection reagent can be used to prepare a kit for detecting 7 pathogens causing spinal infections.

[0009] The present invention realizes the object of the present invention by adopting the following technical solutions:

[0010] 1. Extraction of sample nucleic acid (DNA), and the sample is blood;

[0011] 2. Using the nucleic acid in step 1 as a template, adopting the above specific primers and probes targeting 7 pathogens, and performing detection by multiplex real-time fluorescence quantitative PCR, with the GAPDH gene as an internal reference;

[0012] Specific primers and probes for Staphylococcus aureus, Escherichia coli, Brucella, and Streptococcus agalactiae are used together in the detection; specific primers and probes for Staphylococcus epidermidis, Klebsiella pneumoniae, and Mycobacterium tuberculosis are used together in the detection.

[0013] The amplification reaction system for detecting pathogens using the multiplex fluorescence quantitative PCR detection reagent of the present invention is as follows: 15 μL of 2×SuperRealPreMix (Probe), 0.6 μL each of the upstream and downstream primers and probes for the pathogen, 2 μL each of the DNA templates, and supplemented with ddH2O to 30 μL; the reaction program is: ① template denaturation (95°C, 5 s); ② annealing / extension combination step (55°C, 10 s → 72°C, 30 s), performed for a total of 40 cycles.

[0014] 3. The interpretation of positive test results includes: (1) The Ct value of the internal reference (GAPDH gene) ≤ 36, and there is no Ct value in the negative control group and the no-template control group; if not met, multiple real-time fluorescence quantitative PCR detection must be performed again, or nucleic acid extraction must be repeated for multiple real-time fluorescence quantitative PCR detection; (2) The Ct value of the pathogen ≤ 36.0, if the Ct value > 36.0, single real-time fluorescence quantitative PCR verification needs to be performed for this pathogen; (3) The amplification curve shows a standard "S" shape and has no abnormal fluctuations.

[0015] Compared with the prior art, the present invention has the following advantages and technical effects:

[0016] 1. The primer and probe combination for detecting 7 kinds of spinal infection pathogens provided by the present invention has high detection efficiency, accurate detection results, can complete pathogen diagnosis quickly at low cost, and through specific evaluation of the MqPCR method, it is found that there is no cross-reaction between each group of pathogens and pathogens outside other groups, and the specificity of each group of pathogens is good; through sensitivity evaluation, it is found that except for the detection limits of Staphylococcus aureus (SA), Staphylococcus epidermidis (SE), and Mycobacterium tuberculosis (MTB) being at the order of 10 1 copies / μL, the sensitivities of 4 pathogens and the internal reference GADPH gene all reach the order of 1 copy / μL; through repeatability evaluation of this method, it is found that the coefficient of variation (CV) of each group of pathogens between batches and within batches is less than 2.35%, and the repeatability is good; through MqPCR accuracy evaluation by detecting 10 blood samples, it is found that the MqPCR method has good accuracy;

[0017] 2. Based on the real-time fluorescence quantitative PCR technology platform, the present invention qualitatively detects the specific target genes of pathogens, can detect multiple samples at one time, and has the characteristics of rapidity, specificity, economy, etc., greatly reducing the detection cost of each sample. Description of the Drawings

[0018] Figure 1 The single qPCR specificity test results for SA;

[0019] Figure 2 The single qPCR specificity test results for EC;

[0020] Figure 3 The single qPCR specificity test results for Br;

[0021] Figure 4 The single qPCR specificity test results for SAG;

[0022] Figure 5 The single qPCR specificity test results for SE;

[0023] Figure 6 The single qPCR specificity test results for KP;

[0024] Figure 7 The single qPCR specificity test results for MTB;

[0025] Figure 8 The single qPCR specificity test results for the internal reference GADPH gene;

[0026] Figure 9 The multiplex qPCR specificity test results for SA, EC, Br, SAG and GAPDH;

[0027] Figure 10 The multiplex qPCR specificity test results for SE, MTB, KP and GAPDH;

[0028] Figure 11 The multiplex qPCR sensitivity test results for SA, EC, Br and SAG. In the figure: The sensitivity amplification curves of Staphylococcus aureus (SA) are 1.60×10 7 copies / μL to 1.60×10 1 copies / μL from left to right; The sensitivity amplification curves of Escherichia coli (EC) are 1.63×10 7 copies / μL to 1.63×10 0 copies / μL from left to right; The sensitivity amplification curves of Brucella (Br) are 1.22×10 7 copies / μL to 1.22×10 0 copies / μL from left to right; The sensitivity amplification curves of Streptococcus agalactiae (SAG), with dilution gradients from left to right, are 1.22×10 7 copies / μL to 1.22×10 0 copies / μL;

[0029] Figure 12 The results of the multiplex qPCR sensitivity test for SE, MTB, and KP. In the figure, the sensitivity amplification curves of Staphylococcus epidermidis (SE) are 1.40×10 7 copies / μL to 1.40×10 1 copies / μL from left to right; the sensitivity amplification curves of Klebsiella pneumoniae (KP) are 1.80×10 7 copies / μL to 1.80×10 0 copies / μL from left to right; for the sensitivity amplification curve of Mycobacterium tuberculosis (MTB), the dilution gradient is 1.22×10 7 copies / μL to 1.22×10 1 copies / μL from left to right;

[0030] Figure 13 The results of the multiplex qPCR sensitivity test for the internal reference GADPH gene. In the figure, the sensitivity amplification curves of the first group of internal reference (GAPDH) are 1.50×10 7 copies / μL to 1.50×10 1 copies / μL from left to right; the sensitivity amplification curves of the second group of internal reference (GAPDH) are 1.50×10 7 copies / μL to 1.50×10 0 copies / μL from left to right. Detailed implementation manners

[0031] The technical solutions of the present invention will be further described in detail below through examples. However, the content of the present invention is not limited thereto. In the examples, the methods are conventional methods unless otherwise specified, and the materials, reagents, etc. are obtained from commercial channels or prepared by conventional methods unless otherwise specified;

[0032] Example 1: Design of primers and probes

[0033] 1. Download the pathogen gene reference sequences on the NCBI (National Center for Biotechnology Information) website as follows: The Nuc-encoding gene for Staphylococcus aureus, the 16S-encoding gene for Staphylococcus epidermidis, the ECs104-encoding gene for Escherichia coli, the IS986-encoding gene for Mycobacterium tuberculosis, the HAMP domain-containing sensor histidine kinase-encoding gene for Streptococcus agalactiae, the IS711-encoding gene for Brucella, and the waa-encoding gene for Klebsiella pneumoniae. Align the nucleotide sequences using the Mega 7 software, and design primers and probes using the Primer Select software, and the following conditions need to be met:

[0034] (1) Tm value: Generally, the Tm value of the probe is 8 - 10 °C higher than that of the primer, and the Tm value of the probe is generally above 60 °C;

[0035] (2) GC content: Generally not less than 40%;

[0036] (3) No primer dimers are produced, and the software evaluation result of the hairpin structure is OK;

[0037] (4) The amplified fragment size is generally less than 200 bp;

[0038] 2. BLAST evaluation of primers and probes: For the nucleotide sequences of the primers and probes preliminarily designed, use the BLAST search function on the NCBI website again for alignment, and select the primer and probe sequences with high specificity;

[0039] The nucleotide sequences of the specific primers and probes targeting 7 pathogens of central nervous system infectious diseases and the internal reference GAPDH gene are shown in the following table;

[0040]

[0041]

[0042] 4. Construction of plasmids

[0043] The specific sequences of 7 pathogens (nucleotide sequences as shown in SEQ ID NO: 22 - SEQ ID NO: 28), the sequence of the internal reference gene GAPDH (nucleotide sequence as shown in SEQ ID NO: 29) were ligated to the pUC57 vector to synthesize plasmid standards. One plasmid was constructed for each of Staphylococcus aureus (SA), Escherichia coli (EC), Staphylococcus epidermidis (SE), Klebsiella pneumoniae (KP), and the internal reference gene GAPDH. For Brucella (Br), Streptococcus agalactiae (SAG), and Mycobacterium tuberculosis (MTB) gene clusters, a multi - expression vector system was used and synthesized onto one plasmid. The plasmid construction was completed by General Biotechnology Co., Ltd.; the concentration was determined by ultraviolet spectrophotometer, and the copy number of the plasmid was calculated based on the length and concentration of each plasmid;

[0044]

[0045]

[0046] The above - mentioned plasmids were serially diluted by a 10 - fold dilution method, and a total of seven concentrations were set, which were 10 7 、10 6 、10 5 、10 4 、10 3 、10 2 、10, 1 copies / μL.

[0047] Example 2: qPCR amplification and specificity, sensitivity, and repeatability tests

[0048] 1. Single - plex fluorescence quantitative PCR

[0049]

[0050] 10 μL of plasmid standards with a concentration of 10 3 copies / μL were taken and mixed as templates, and a single - plex fluorescence quantitative PCR detection was performed using the SuperReal fluorescence quantitative premixed probe method qPCR kit from Tiangen Biotech Co., Ltd. The mixed plasmid templates of 7 pathogens were detected with the specific primers and probes of each pathogen respectively. The detection results are as Figures 1-8 shown; it can be seen from the results that amplification curves were obtained for all 7 pathogens and the internal reference gene.

[0051] 2. Multiplex qPCR specificity test

[0052] The detection of 7 pathogens was carried out in 2 groups. Among them, Staphylococcus aureus, Escherichia coli, Brucella, Streptococcus agalactiae, and the internal reference gene were in one group; Staphylococcus epidermidis, Klebsiella pneumoniae, Mycobacterium tuberculosis, and the internal reference gene were in the other group;

[0053] The multiplex fluorescence quantitative PCR reaction system is 30 μL, in which 0.6 μL of primers for 4 pathogens and 1 internal reference gene is added, 0.4 μL of probe is added, and the template is 2 μL:

[0054]

[0055] The amplification reaction procedure is as follows: template denaturation (95 °C, 5 s); annealing / extension combination step (55 °C, 10 s → 72 °C, 30 s), which is performed for 40 cycles, and fluorescence signals are collected during the annealing and extension stage of each cycle.

[0056] Take 10 μL of plasmid standards with a concentration of 10 3 copies / μL and mix them as templates respectively. Use the SuperReal fluorescence quantitative premixed probe method qPCR kit from Tiangen Biotech Company to perform multiplex fluorescence quantitative PCR detection. Detect the mixed plasmid templates of 7 pathogens using specific primers and probes according to the above grouping. The results are as Figures 9-10 ; It can be seen from the above results that there is no cross-reaction among the pathogens in each group, indicating that the multiplex fluorescence quantitative PCR has good specificity.

[0057] 3. Multiplex qPCR sensitivity test

[0058] Use the MqPCR method to detect plasmid templates with gradients of 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 , 1 copies / μL to determine the lowest plasmid concentration that the MqPCR detection method can detect. The results are as Figures 11-13 shown. It can be seen from Figure 11 that for SA, EC, Br, and SAG, the lower limits of sensitivity detection are: 1.63×10 0 , 1.22×10 0 , 1.22×10 0 , 1.80×10 0 copies / μL; It can be seen from Figure 12 that for SE, MTB, and KP, the lower limits of sensitivity detection are: 1.60×101, 1.40×101, 1.22×10 1 copies / μL; It can be seen from Figure 13 the results of the multiplex qPCR sensitivity test of the GADPH gene. The detection limits of GAPDH in the two groups are 1×10 1 , 1 copies / μL level.

[0059] 4. Multiple qPCR Repeatability Test

[0060] Select a standard quality plasmid with a concentration of 10 3 copies / μL to perform a multiple qPCR experiment to evaluate the repeatability of primers and probes in the multiple qPCR system. Use a pathogenic plasmid with a concentration of 10 3 copies / μL as a template, set up 3 groups of parallel experiments, each experiment is repeated three times, lasting for three weeks, and an experiment is carried out at a fixed time every week. The experiment uses a SuperReal fluorescent quantitative premixed probe method qPCR kit. Each group of pathogens and internal reference plasmids are added to the multiple reaction system with the corresponding primers and probes, and RNase-free H2O is used for double blank control. Record the Ct values of each experiment to evaluate the repeatability of the primers and probes. The repeatability results are shown in the following table:

[0061]

[0062] It can be seen from the results in the table that the coefficient of variation (CV) of each group of pathogens between batches and within batches is less than or equal to 2.35%, and the repeatability is good.

[0063] Example 3: Detection of Blood Samples

[0064] 1. Sample Collection

[0065] For the collection of venous blood samples, the elbow vein of the upper arm is usually selected. First, the medical staff disinfect the blood collection site and use a tourniquet to help the vein appear, and then insert a sterile needle into the vein to draw blood samples. The blood is collected in a test tube containing the anticoagulant EDTA. After collection, mark the patient information and store it in a -20°C refrigerator for laboratory testing.

[0066] 2. Genomic DNA Extraction

[0067] DNA Extraction from Blood Samples:

[0068] (1) Sample Pretreatment: Transfer 200 μL of blood sample to a 1.5 mL centrifuge tube, add 20 μL of Proteinase K, mix well, then add 200 μL of binding solution BD, immediately shake well, centrifuge briefly and collect the liquid on the tube wall;

[0069] (2) Incubation: Place the sample in a 70°C water bath for 10 min. After taking it out and allowing it to cool to room temperature, add 100 μL of isopropanol and shake vigorously for 15 s;

[0070] (3) Transfer: Add the pretreated mixture to the DNA adsorption column KP treated with buffer AC, centrifuge at 12000 rpm for 1 min, and discard the waste liquid;

[0071] (4) Deproteinization treatment: Add 500 μL of deproteinization solution PL, centrifuge at 12,000 rpm for 30 s, and discard the waste liquid;

[0072] (5) Rinsing: Add 600 μL of rinsing solution W, centrifuge at 12,000 rpm at room temperature for 30 s, discard the waste liquid, (repeat rinsing twice);

[0073] (6) Removal of residual rinsing solution: Centrifuge the empty column at 12,000 rpm at room temperature for 2 min to remove the residual rinsing solution W;

[0074] (7) Elution: Place the DNA adsorption column B2 into a new 1.5 mL centrifuge tube, add 50 μL of elution solution to the center of the adsorption column, let it stand at room temperature for 2 min, then centrifuge at 12,000 rpm for 1 min to collect the filtrate. Again, load the filtrate onto the column, let it stand at room temperature for 2 min, elute and collect the DNA solution.

[0075] 3. Collect 10 blood samples from the hospital, perform bacterial culture to determine the types of pathogens, and at the same time use the MqPCR method of the present invention to detect these 10 samples, record the detection results, and the results are as follows:

[0076]

[0077] As can be seen from the above table, the primer and probe set for multiplex qPCR detection of the 7 main exogenous pathogens of spinal infection designed by the present invention is faster and more convenient than the detection method of bacterial culture.

Claims

1. A multiplex qPCR detection reagent for detecting spinal infection pathogens, characterized in that: Including specific primers and probes for detecting Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Mycobacterium tuberculosis, Brucella, Klebsiella pneumoniae, and Streptococcus agalactiae; The specific primers are SEQ ID NO:1 and SEQ ID NO:2 for Staphylococcus aureus, SEQ ID NO:4 and SEQ ID NO:5 for Staphylococcus epidermidis, SEQ ID NO:7 and SEQ ID NO:8 for Escherichia coli, SEQ ID NO:10 and SEQ ID NO:11 for Mycobacterium tuberculosis, SEQ ID NO:13 and SEQ ID NO:14 for Brucella, SEQ ID NO:16 and SEQ ID NO:17 for Klebsiella pneumoniae, and SEQ ID NO:19 and SEQ ID NO:20 for Streptococcus agalactiae; The probes are SEQ ID NO:3 for Staphylococcus aureus, SEQ ID NO:6 for Staphylococcus epidermidis, SEQ ID NO:9 for Escherichia coli, SEQ ID NO:12 for Mycobacterium tuberculosis, SEQ ID NO:15 for Brucella, SEQ ID NO:18 for Klebsiella pneumoniae, and SEQ ID NO:21 for Streptococcus agalactiae.

2. Use of the multiplex qPCR detection reagent for detecting spinal infection pathogens according to claim 1 in the preparation of a kit for detecting spinal infection pathogens.

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