Primer pair, probe, method and kit for detecting carbapenem drug-resistant acinetobacter baumannii

Abstract

The invention discloses a primer pair and a probe for detecting carbapenem drug-resistant acinetobacter baumannii. The primer pair comprises a specific primer pair and a specific probe of a specific gene 16SRNA, and a drug-resistant primer pair and a drug-resistant probe of a drug-resistant gene OXA-23, an upstream primer sequence of the specific primer pair is as shown in SEQ ID NO: 1, a downstream primer sequence is as shown in SEQ ID NO: 2, and a specific probe sequence is as shown in SEQ ID NO: 3; an upstream primer sequence of the drug-resistant primer pair is as shown in SEQ ID NO: 4, a downstream primer sequence of the drug-resistant primer pair is as shown in SEQ ID NO: 5, and a probe sequence is as shown in SEQ ID NO: 6; according to the primer pair, the probe, the method and the kit provided by the invention, corresponding primers and probes are designed and optimized according to 16SRNA and OXA-23 genes, and through construction of a 16SRNA / OXA-23 double qPCR amplification reaction system, acinetobacter baumannii in blood flow infection can be rapidly and accurately detected, and whether carbapenem drug resistance exists or not is judged; and clinical timely diagnosis and accurate treatment are helped.

Description

Technical Field

[0001] The present invention relates to the field of gene detection technology, and specifically relates to primer pairs and probes, methods and kits for detecting carbapenem-resistant Acinetobacter baumannii. Background Art

[0002] Bloodstream infection (BSI) is a serious life-threatening systemic infectious disease, especially bloodstream infection caused by multidrug-resistant bacteria (such as carbapenem-resistant Gram-negative bacilli), with a high mortality rate. Acinetobacter baumannii (AB) is a non-fermentative conditional pathogenic Gram-negative bacillus and is one of the common pathogens causing hospital-acquired infections. The key to treating bloodstream infection is the timely and accurate diagnosis of pathogens and the effective use of antibiotics. However, Acinetobacter baumannii has brought many difficulties to the treatment of bloodstream infection due to its extensive drug resistance, which seriously endangers the lives of patients.

[0003] Carbapenem antibiotics include imipenem, meropenem and other drugs. They are one of the first-line drugs for empirical treatment of bloodstream infection and are regarded as the last line of defense for the treatment of Acinetobacter baumannii infection. The emergence of drug resistance is the main cause of death after bloodstream infection with Acinetobacter baumannii. The main mechanism of Acinetobacter baumannii resistance to carbapenem antibiotics is carbapenemases, including class B metallo-β-lactamases and class D oxacillinase (OXA), of which OXA-23 is one of the most common genes for carbapenem resistance in Acinetobacter baumannii.

[0004] At present, the gold standard for diagnosing bloodstream infection is still positive blood culture (BC). Relevant statistics show that it takes about 6 hours to 5 days for clinical patients to culture venous blood to a level that can be detected by the instrument. After the instrument reports a positive blood culture, it takes one day to culture a certain amount of single colonies on the plate for identification. After identification, it takes at least one day to perform drug sensitivity identification. Therefore, it takes at least 3 days from collecting the specimen to the final drug sensitivity report. Data show that in patients with sepsis within 6 hours before the recorded hypotension, every 1 hour delay in appropriate antibiotic treatment will lead to an average increase of 7.6% in mortality. Timely and accurate identification of pathogens and selection of appropriate antimicrobial drugs are crucial to the treatment and prognosis of patients with early bloodstream infection. Therefore, it is urgent to develop new technologies for rapid diagnosis of bloodstream infection.

[0005] The technologies currently under study for diagnosing bloodstream infections include metagenomic next-generation sequencing (mNGS), droplet digital PCR (ddPCR) and fluorescence quantitative PCR technology. The former two are relatively expensive, so this application establishes a dual qPCR reaction system to detect Acinetobacter baumannii and its carbapenem resistance in bloodstream infections, thereby improving the sensitivity and specificity of qPCR in detecting carbapenem-resistant Acinetobacter baumannii DNA. Summary of the invention

[0006] To this end, the technical problem to be solved by the present invention is to provide a primer pair and probe, method and kit for detecting carbapenem-resistant Acinetobacter baumannii. The corresponding primers and probes are designed and optimized according to the 16SRNA and OXA-23 genes. By constructing a 16SRNA / OXA-23 dual qPCR amplification reaction system, Acinetobacter baumannii in hemorrhage infection can be quickly and accurately detected and whether it has carbapenem resistance, thereby helping clinical timely diagnosis and precise treatment.

[0007] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0008] A primer pair and probe for detecting carbapenem-resistant Acinetobacter baumannii, characterized in that it comprises a specific primer pair for the specific gene 16SRNA and a specific probe matched to the specific primer pair, and a drug-resistant primer pair for the drug-resistant gene OXA-23 and a drug-resistant probe matched to the drug-resistant primer pair;

[0009] The upstream primer sequence of the specific primer pair is shown in SEQ ID NO: 1, which is 5'-AAG CGA GGA GGAGGC TAC TTT AG-3';

[0010] The downstream primer sequence of the primer of the specific primer pair is shown in SEQ ID NO: 2, which is 5'-CGG CTGCTG GCA CAG AGT-3';

[0011] The specific probe sequence is shown in SEQ ID NO: 3, which is 5-ACG TTA CTC GCA GAA TAA GCACCG GCT-3;

[0012] The upstream primer sequence of the drug-resistant primer pair is shown in SEQ ID NO: 4, which is 5'-GAC ACT AGG AGAAGC CAT GAA GCT-3';

[0013] The downstream primer sequence of the drug-resistant primer pair is shown in SEQ ID NO: 5, which is 5'-GCA TGA GAT CAAGAC CGA TAC G-3';

[0014] The drug-resistant probe sequence is shown in SEQ ID NO: 6, which is 5-TCC CAG TCT ATC AGG AAC TTG CGCG-3.

[0015] Furthermore, the concentration of the specific primer pair is 300-600 nM, the concentration of the drug-resistant primer pair is 300-600 nM, and the concentration ratio of the specific primer pair to the drug-resistant primer pair is 500-600 nM:300-500 nM.

[0016] A kit for detecting carbapenem-resistant Acinetobacter baumannii, comprising a specific primer pair and a specific probe combination of a specific gene 16SRNA and a resistance primer pair and a resistance probe combination of a resistance gene OXA-23:

[0017] The upstream primer sequence of the specific primer pair is shown in SEQ ID NO: 1, the downstream primer sequence of the primer of the specific primer pair is shown in SEQ ID NO: 2, and the specific probe sequence is shown in SEQ ID NO: 3;

[0018] The upstream primer sequence of the drug-resistant primer pair is shown in SEQ ID NO: 4, the downstream primer sequence of the drug-resistant primer pair is shown in SEQ ID NO: 5, and the drug-resistant probe sequence is shown in SEQ ID NO: 6.

[0019] Furthermore, the kit is used in the preparation of drugs for diseases caused by carbapenem-resistant Acinetobacter baumannii containing the resistance gene OXA-23.

[0020] Furthermore, the kit is used for a qPCR amplification reaction system, wherein a 20 μL reaction system comprises:

[0021] CR mix 10 μL, 10 μM primer pairs and probes;

[0022] Primer pairs, 1 μL each of the upstream primer and downstream primer of the specific primer pair and 1 μL each of the upstream primer and downstream primer of the drug-resistant primer pair, and probes including 0.5 μL of the specific probe and 0.5 μL of the drug-resistant probe;

[0023] 2 μL of DNA template;

[0024] Add distilled water to make up to 20 μL.

[0025] A method for detecting carbapenem-resistant Acinetobacter baumannii, comprising a specific primer pair for a specific gene 16SRNA and a specific probe matched to the specific primer pair, and a drug-resistant primer pair for a drug-resistant gene OXA-23 and a drug-resistant probe matched to the drug-resistant primer pair;

[0026] The upstream primer sequence of the specific primer pair is shown in SEQ ID NO: 1, the downstream primer sequence of the primer of the specific primer pair is shown in SEQ ID NO: 2, and the specific probe sequence is shown in SEQ ID NO: 3;

[0027] The upstream primer sequence of the drug-resistant primer pair is shown in SEQ ID NO: 4, the downstream primer sequence of the drug-resistant primer pair is shown in SEQ ID NO: 5, and the drug-resistant probe sequence is shown in SEQ ID NO: 6;

[0028] The steps include:

[0029] S1. Extract DNA from the sample to be tested;

[0030] S2. Using the extracted DNA as a template, a duplex qPCR reaction system with a total volume of 20 μL was constructed, and specific primer pairs and specific probes at a concentration of 10 μM as well as drug-resistant primer pairs and drug-resistant probes were added to perform qPCR amplification reactions;

[0031] S3. Verify the detection limit and linear relationship of 16sRNA and OXA-23 in the dual qPCR reaction system.

[0032] Furthermore, in step S2, the qPCR amplification step is: 39 cycles are performed in the order of 95°C pre-denaturation for 30 seconds - 95°C denaturation for 5 seconds - 60°C annealing for 30 seconds.

[0033] Furthermore, the annealing temperature of the dual qPCR reaction system is 57.3°C-58.4°C.

[0034] The technical solution of the present invention has achieved the following beneficial technical effects

[0035] Adding the corresponding primers of the two genes 16SRNA and OXA-23 to the same reaction system can effectively distinguish Acinetobacter baumannii from other 26 pathogens. While detecting Acinetobacter baumannii, its carbapenem resistance can be determined, and two gene fragments can be amplified at the same time, reflecting the advantages of high efficiency and low cost of dual qPCR. It has high sensitivity, strong specificity and a wide detection range, and can also quickly and accurately detect CRAB in bloodstream infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 A schematic diagram of the results of constructing a dual qPCR reaction system of primer pairs and probes, methods and kits for detecting carbapenem-resistant Acinetobacter baumannii according to the present invention;

[0037] A is the CSAB reaction result of the dual qPCR reaction system, B is the CRAB reaction result of the dual qPCR reaction system, the green curve in the figure represents 16SRNA, the blue curve represents OXA-23, 1 in A is a negative control, 2 and 3 are CSAB, 4 in B is a negative control, 5, 6, 7 are CRAB);

[0038] Figure 2 Schematic diagram of optimization results of the dual qPCR reaction system of the present invention;

[0039] A is the optimization result of 16SRNA primer concentration; B is the optimization result of OXA-23 primer concentration; primer concentration ratio optimization result; C and D are the optimization results of concentration ratio, where green represents a concentration ratio of 500nM:400nM, orange represents a concentration ratio of 500nM:500nM, and blue represents a concentration ratio of 600nM:500nM; red represents a concentration ratio of 500nM:300nM, and purple represents a concentration ratio of 600nM:400nM;

[0040] Figure 3 Schematic diagram of the specificity verification results of the present invention;

[0041] A is the verification result of hepatitis B virus, Candida albicans, Candida tropicalis, Candida glabrata, Escherichia coli, and Klebsiella pneumoniae;

[0042] B is the verification results of Staphylococcus epidermidis, Staphylococcus aureus, Staphylococcus capitis, Staphylococcus hominis, Staphylococcus haemolyticus, and Streptococcus pneumoniae;

[0043] C is the verification results of Enterococcus faecium, Enterococcus faecalis, Pseudomonas aeruginosa, Enterobacter cloacae, Proteus mirabilis, and Propionibacterium acnes;

[0044] D is the verification result of Stenotrophomonas maltophilia, Streptococcus mitis, Staphylococcus lugdunensis, Klebsiella oxytoca, Corynebacterium taeniae, Serratia marcescens, Staphylococcus worderi, and Morganella morganii;

[0045] The green curve is the 16SRNA positive control, the blue curve is the OXA-23 positive control, and the curve below the threshold line is a negative result;

[0046] Figure 4 Schematic diagram of sensitivity experiment results of the present invention;

[0047] A is the result of 16S RNA, B is the result of OXA-23, and 1-7 are the DNA concentrations of 3×10 2 -3×10 -4 ng / μL experimental results; C is the 16sRNA standard curve, and D is the OXA-23 standard curve. DETAILED DESCRIPTION

[0048] The present invention will be further described below with reference to specific embodiments, but is not limited thereto.

[0049] Example 1

[0050] The primer pairs and probes for detecting carbapenem-resistant Acinetobacter baumannii include a specific primer pair for the specific gene 16SRNA and a specific probe matched with the specific primer pair, as well as a drug-resistant primer pair for the drug-resistant gene OXA-23 and a drug-resistant probe matched with the drug-resistant primer pair.

[0051] By searching the gene sequences of 16sRNA and OXA-23 in the NCB I database, relevant primers and probes were designed and optimized for these two sequences.

[0052] The nucleotide sequences of the primers for the specific gene 16SRNA are as follows:

[0053] Upstream primer: 5′-AAG CGA GGA GGA GGC TAC TTT AG-3′ (SEQ ID NO: 1);

[0054] Downstream primer: 5′-CGG CTG CTG GCA CAG AGT-3′ (SEQ ID NO: 2);

[0055] Probe: 5-ACG TTA CTC GCA GAA TAA GCA CCG GCT-3 (SEQ ID NO: 3);

[0056] The primer nucleotide sequence of the drug resistance gene OXA-23 is as follows:

[0057] Upstream primer: 5′-GAC ACT AGG AGA AGC CAT GAA GCT-3′ (SEQ ID NO: 4);

[0058] Downstream primer: 5'-GCA TGA GAT CAA GAC CGA TAC G-3' (SEQ ID NO: 5);

[0059] Probe: 5-TCC CAG TCT ATC AGG AAC TTG CGC G-3 (SEQ ID NO: 6).

[0060] Targeting the 16SRNA and OXA-23 genes, adding primers corresponding to the two genes into the same reaction system can effectively distinguish Acinetobacter baumannii from other 26 pathogens. While detecting Acinetobacter baumannii, its carbapenem resistance can be determined. The simultaneous amplification of two gene fragments demonstrates the advantages of dual qPCR in terms of high efficiency and low cost. In addition, qPCR has relatively high sensitivity and specificity in detecting microbial DNA.

[0061] Example 2

[0062] Clinically isolated blood cultures were collected to detect strains of Acinetobacter baumannii and other microorganisms including fungi, bacteria, and viruses. After the corresponding DNA was extracted by column extraction, a double reaction system was established for the drug-resistant gene OXA-23 and the Acinetobacter baumannii-specific gene 16sRNA using fluorescent quantitative PCR. At the same time, specificity, sensitivity, linearity experiments, repeatability and clinical sample verification were performed. The specific experimental process is as follows.

[0063] 1. Experimental Materials

[0064] 1.1 Specimen source

[0065] The samples used in this experiment were all from 7 strains of CSAB, 15 strains of CRAB and 26 other different types of bacteria, fungi, viruses, etc. isolated from positive clinical blood cultures in the Microbiology Laboratory of the First Affiliated Hospital of Guangzhou Medical University from July 2022 to April 2024. The collected samples were isolated, cultured and identified according to the "National Clinical Laboratory Operation Procedure" (Fourth Edition).

[0066] 1.2 Main instruments and reagents

[0067] The fluorescence quantitative PCR instrument was produced by Bio-rad, USA, the vortex mixer was produced by Qilin Bell, Haimen, China, the Nano Drop Vue Plus ultra-micro-volume spectrophotometer was produced by GE, USA, and the QIAamp DNA purification min kit was produced by Shanghai Qiagen Enterprise Management Co., Ltd.

[0068] 2. Experimental methods

[0069] 2.1 Collection and preservation of strains

[0070] Acinetobacter baumannii isolated and cultured from clinical positive blood cultures and other bacteria and fungi that are positive for blood cultures were collected. The corresponding colonies on blood agar plates, chocolate plates or fungal selection culture plates identified by mass spectrometry were collected into preservation tubes, numbered and stored in a -80°C refrigerator for future use.

[0071] 2.2 Extraction of bacterial DNA

[0072] Bacterial DNA was extracted using the column extraction method. The strains were thawed and separated on blood agar plates. The strains were cultured in a 37°C incubator for 8 to 24 hours. DNA was extracted using a bacterial extraction kit. The DNA concentration and purity were detected using NanoDrop2000 and finally stored in a -20°C refrigerator. Bacterial DNA in venous blood samples was extracted using QIAamp DNA Mini Kit (50) reagent (QIAGEN). The DNA concentration and purity were measured and recorded and stored in a -20°C refrigerator.

[0073] 2.3 Construction of dual qPCR reaction system

[0074] A duplex qPCR reaction system with a total volume of 20 μL was designed, into which primers and probes with a concentration of 10 μM were added, including 10 μL qPCR mix, 1 μL each of the upstream and downstream primers of 16sRNA / OXA-23, 0.5 μL each of the 16sRNA / OXA-23 probe, and 2 μL of DNA. The total volume was made up to 20 μL with distilled water. According to the instructions of the Probe qPCR Mix reagent, the qPCR amplification steps were as follows: pre-denaturation at 95°C for 30 seconds; denaturation at 95°C for 5 seconds; annealing at 60°C for 30 seconds; and 39 cycles in total.

[0075] Sterile water was used as negative control, and different CRAB and CSAB were used to verify the feasibility of dual qPCR reaction of primers and probes with different CRAB and CSAB.

[0076] 2.4 Optimization of the dual qPCR reaction system

[0077] The amplification efficiency of the qPCR reaction system is related to factors such as the concentration of the primer probe, the ratio between the two primers, and the annealing temperature. These factors have a significant impact on the results by affecting factors such as the Ct value and fluorescence intensity. This experiment optimized the three variables of primer concentration, primer concentration ratio, and annealing temperature.

[0078] 2.4.1 Optimization of primer concentrations for dual qPCR

[0079] The ratio of 16sRNA / OXA-23 was fixed at 1:1. In the constructed dual qPCR reaction system, the optimized final concentrations of primers and probes were selected as: 600nM, 500nM, 400nM, and 300nM. The probe concentrations were adjusted according to the primer-probe ratio of 2:1. The optimal primer concentration was determined according to the Ct value and fluorescence intensity.

[0080] 2.4.2 Optimization of the 16sRNA / OXA-23 Primer-Probe Concentration Ratio for Dual qPCR

[0081] In order to explore whether there is an inhibitory and interfering effect between the two primers in the dual qPCR reaction system, 16sRNA and OXA-23 were added to the reaction system in different ratios for amplification reaction. The final concentrations of 16sRNA / OXA-23 were selected as follows: 300nM:500nM, 400nM:500nM, 500nM:500nM, 500nM:600nM, 400nM:600nM, and the concentration of the probe was adjusted according to the ratio of primer to probe of 2:1. The optimal primer concentration ratio was determined according to the Ct value and fluorescence intensity.

[0082] 2.4.3 Optimization of duplex qPCR annealing temperature

[0083] According to the optimal primer concentration and primer concentration ratio determined in the above experiment, the annealing temperature was optimized. In this experiment, 65°C, 64.5°C, 63.4°C, 61.9°C, 60°C, 58.4°C, 57.3°C, and 56.7°C were selected as annealing temperatures for amplification. The optimal annealing temperature was determined based on the Ct value and fluorescence intensity.

[0084] 2.5 Duplex qPCR specificity verification

[0085] Water blank, CSAB, CRAB and 26 clinically common bacteria, fungi and viral DNA shown in Figure 1 were selected to detect the specificity of the corresponding primer pairs and probes, including Escherichia coli, Klebsiella pneumoniae, Staphylococcus epidermidis, Staphylococcus aureus, Staphylococcus capitis, Pseudomonas aeruginosa, Enterococcus faecium, Enterococcus faecalis, Staphylococcus hominis, Staphylococcus haemolyticus, Enterobacter cloacae, Streptococcus pneumoniae, Proteus mirabilis, Propionibacterium acnes, Stenotrophomonas maltophilia, Streptococcus mitis, Staphylococcus lugdunensis, Klebsiella oxytoca, Corynebacterium striatum, Serratia marcescens, Staphylococcus voriconii, Morganella morganii, hepatitis B virus, Candida albicans, Candida tropicalis, Candida glabrata, etc.

[0086] Table 1 26 fungi, bacteria, and viruses with verified specificity

[0087]

[0088]

[0089] 2.6 Duplex qPCR limit of detection (LOD) and linearity experiments

[0090] From the extracted DNA of drug-resistant Acinetobacter baumannii, a 3×10 2 ng / μl of DNA, and then the DNA was diluted in multiples to a concentration of 3×10 2 , 3×10 1 , 3×100 , 3×10 -1 , 3×10 -2 , 3×10 -3 , 3×10 -4 ng / μL, sterile water was used as negative control, and each diluted concentration was added as DNA to the dual qPCR reaction system. Two tubes were set for each concentration for the experiment. The sensitivity was determined based on whether a specific curve could be amplified. Finally, the average of the two Ct value results for each concentration within the detection range was calculated. The logarithm of the theoretical concentration was used as the horizontal axis and the Ct value as the vertical axis. The coordinate axis was established, the standard curve was made, the correlation coefficient was obtained, and the linearity of the curve was determined.

[0091] 2.7 Duplex qPCR reproducibility experiment

[0092] According to the sensitivity results, DNA concentrations within the linear range were selected for repeatability experiments. The experiment was repeated three times for each tube of DNA. The Ct values ​​of the three results were calculated to calculate the mean, standard deviation, and coefficient of variation to evaluate the repeatability and stability of the method.

[0093] 2.8 Clinical Validation

[0094] Venous blood samples were collected from patients with positive clinical blood culture, and bacterial DNA was extracted using the QIAamp DNA purification mini kit. Dual qPCR experiments were then performed to determine whether this method could specifically detect Acinetobacter baumannii in patients' bloodstream infections and whether it was carbapenem-resistant.

[0095] 3. Experimental results

[0096] 3.1 Construction of dual qPCR reaction system

[0097] Using sterile water as negative control, CSAB and different CRAB were selected to construct a dual qPCR system. The results are shown in Figure 1 As shown in the figure, the fluorescence channel of the 16S RNA probe designed in this experiment is HEX, and the fluorescence channel of the OXA-23 probe is FAM. Only 16S RNA of CSAB produces fluorescence, while both 16S RNA and OXA-23 of CRAB produce fluorescence, which is a positive result. This shows that the established dual qPCR system can detect Acinetobacter baumannii and determine its resistance to carbapenems.

[0098] 3.2 Optimization of the Dual qPCR Reaction System

[0099] 3.2.1 Optimization of primer concentration for dual qPCR

[0100] To verify the effect of primer concentration on amplification, four primer concentrations of 300nM, 400nM, 500nM, and 600nM were set according to the commonly used range of primer concentrations for optimization experiments. The results are as follows: Figure 2 As shown in A and 2B, the Ct values ​​of the four concentrations remained basically unchanged. The primer concentration had a certain effect on the Ct value of 16SRNA and OXA-23 amplification. The Ct value decreased with the increase of primer probe concentration, but the change was very small, and the fluorescence intensity increased. Considering that the fluorescence background should be reduced as much as possible while improving the experimental sensitivity, 500nM was used as the optimal primer concentration.

[0101] 3.2.2 Optimization of primer-probe concentration ratio for dual qPCR 16S RNA / OXA-23

[0102] The 16SRNA / OXA-23 primer concentration ratios were 500nM:300nM, 500nM:400nM, 500nM:500nM, 600nM:500nM, 600nM:400nM. Figure 2 The results of C and 2D show that there is no obvious promotion or inhibition relationship. Based on the Ct value and fluorescence intensity, the 16SRNA / OXA-23 primer concentration ratio of 500nM:500nM was selected as the final primer concentration ratio.

[0103] 3.2.3 Optimization of duplex qPCR annealing temperature

[0104] The optimization results of annealing temperature are shown in Table 2. When the annealing temperature decreases with 65℃, 64.5℃, 63.4℃, 61.9℃, 60℃, and 58.4℃, the Ct value decreases accordingly, and the fluorescence intensity increases. When it continues to decrease to 57.3℃ or 56.7℃, the Ct value begins to rise and the fluorescence intensity also decreases. The best result for 16SRNA is at 58.4℃, and the best results for OXA-23 are at 58.4℃ and 57.3℃, so 58.4℃ is selected as the annealing temperature for this experiment.

[0105] Table 2 Annealing temperature optimization results

[0106]

[0107] 3.3 Specificity verification

[0108] The primer probes of qPCR designed in the present invention can specifically detect Acinetobacter baumannii and can also distinguish drug-resistant strains well. The specific verification results of 22 bacteria, 3 fungi and 1 virus are as follows: Figure 3 Resistant Acinetobacter baumannii was used as the positive control and sterile water as the negative control. The specificity verification results showed that no fluorescence was produced within 40 cycles for non-Acinetobacter baumannii, and no S-shaped curve was amplified, which was a negative result, proving that the specific primer pair had good specificity.

[0109] 3.4 Duplex qPCR detection limit and linearity experiments

[0110] Dual qPCR sensitivity results are as follows Figure 4 As shown in A and 4B, when the DNA concentration was diluted to 3×10 -4 ng / μL, there was no fluorescence generation and no Ct value for both 16sRNA and OXA-23 genes, so the minimum detection limit of both 16sRNA and OXA-23 by dual qPCR was 3×10 -3 ng / μL, when the DNA concentration of Acinetobacter baumannii was 3×10 2 -3×10 -3 Positive results can be obtained in the concentration range of ng / μL. The average of the two Ct value results for each concentration is calculated and then the following is done: Figure 4 The standard curves shown in C and 4D, the standard curves of 16SRNA and OXA-23 are y = -4.1329x + 22.832, R2 = 0.9945 and y = -4.0243x + 22.123, R2 = 0.9945, respectively. Therefore, the dual qPCR method has a good linear relationship and a wide linear range for detecting 16SRNA and OXA-23.

[0111] 3.5 Duplex qPCR reproducibility experiment

[0112] According to the sensitivity and linearity test results, 3×10 2 -3×10 -3 ng / μL, representing high, medium and low concentration ranges, respectively, and each concentration was repeated 3 times. The mean of the Ct value of the 3 results for each concentration was calculated, and the standard deviation and coefficient of variation were calculated. It can be seen from Table 3 that the coefficient of variation of each concentration is less than or equal to 2%, which has good repeatability.

[0113] Table 3 Duplex qPCR repeatability experiment

[0114]

[0115]

[0116] 3.6 Clinical Validation

[0117] Venous blood specimens from patients with positive clinical blood culture were collected at the same time, and DNA was extracted using the QIAamp DNA purification kit. The established dual qPCR system was used for clinical specimen verification.

[0118] A total of 30 venous blood specimens were collected at the same time as the microbial venous blood culture, of which 9 were blood culture-negative samples, 6 were blood culture-positive but non-Acinetobacter baumannii samples, and 15 were CSAB and CRAB samples. These samples were subjected to qPCR and drug sensitivity tests as shown in Table 4. The qPCR test of the 30 venous blood samples showed 100% sensitivity and specificity (the positive standard was Ct value <38).

[0119] Table 6 Clinical validation of qPCR assay

[0120]

[0121] In summary, the method established by the present invention has good specificity, high repeatability and is more widely used and practical in clinical practice. Dual qPCR adds two pairs of primers in the same reaction system to amplify two gene fragments at the same time. It can effectively distinguish Acinetobacter baumannii from other 26 pathogens by targeting the two genes 16SRNA and OXA-23. It can detect Acinetobacter baumannii and determine its carbapenem resistance at the same time. Its minimum detection limit is as low as 3×10 -3 ng / μL, with a wide linear range of 3×10 2 -3×10 -3 Positive results can be obtained in the concentration range of ng / μL, and it can be used for the detection of CRAB in clinical bloodstream infection specimens. The results are consistent with the results of blood culture and drug sensitivity, which further demonstrates the advantages of dual qPCR in terms of high efficiency and low cost. In addition, qPCR has relatively high sensitivity and specificity in detecting microbial DNA.

[0122] The present invention is a dual real-time fluorescence quantitative PCR detection method based on 16SRNA and OXA-23, which not only has high sensitivity, strong specificity and a wide detection range, but can also quickly and accurately detect CRAB in bloodstream infection, so it has broad application prospects in the diagnosis of bloodstream infection.

[0123] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the claims of this patent application.

Claims

1. A primer pair and a probe for detecting carbapenem-resistant Acinetobacter baumannii, characterized in that: It includes a specific primer pair of the specific gene 16SRNA and a specific probe matched with the specific primer pair, and a drug-resistant primer pair of the drug-resistant gene OXA-23 and a drug-resistant probe matched with the drug-resistant primer pair; The upstream primer sequence of the specific primer pair is shown in SEQ ID NO: 1, the downstream primer sequence of the primer of the specific primer pair is shown in SEQ ID NO: 2, and the specific probe sequence is shown in SEQ ID NO: 3; The upstream primer sequence of the drug-resistant primer pair is shown in SEQ ID NO: 4, the downstream primer sequence of the drug-resistant primer pair is shown in SEQ ID NO: 5, and the drug-resistant probe sequence is shown in SEQ ID NO:

6.

2. The primer pair and probe according to claim 1, characterized in that: The concentration of the specific primer pair is 300-600 nM, the concentration of the drug-resistant primer pair is 300-600 nM, and the concentration ratio of the specific primer pair to the drug-resistant primer pair is 500-600 nM:300-500 nM.

3. A kit for detecting carbapenem-resistant Acinetobacter baumannii, characterized in that: Including specific primer pairs and specific probe combinations of the specific gene 16SRNA and drug-resistant primer pairs and drug-resistant probe combinations of the drug-resistant gene OXA-23: The upstream primer sequence of the specific primer pair is shown in SEQ ID NO: 1, the downstream primer sequence of the primer of the specific primer pair is shown in SEQ ID NO: 2, and the specific probe sequence is shown in SEQ ID NO: 3; The upstream primer sequence of the drug-resistant primer pair is shown in SEQ ID NO: 4, the downstream primer sequence of the drug-resistant primer pair is shown in SEQ ID NO: 5, and the drug-resistant probe sequence is shown in SEQ ID NO:

6.

4. The kit according to claim 3, characterized in that The kit is used in the preparation of medicines for diseases caused by carbapenem-resistant Acinetobacter baumannii containing the drug resistance gene OXA-23.

5. The kit according to claim 3, characterized in that The kit is used for a qPCR amplification reaction system, wherein a 20 μL reaction system includes: CR mix 10 μL, 10 μM primer pairs and probes; Primer pairs, 1 μL each of the upstream primer and downstream primer of the specific primer pair and 1 μL each of the upstream primer and downstream primer of the drug-resistant primer pair, and probes including 0.5 μL of the specific probe and 0.5 μL of the drug-resistant probe; 2 μL of DNA template; Add distilled water to make up to 20 μL.

6. A method for detecting carbapenem-resistant Acinetobacter baumannii, characterized in that: It includes a specific primer pair of the specific gene 16SRNA and a specific probe matched with the specific primer pair, and a drug-resistant primer pair of the drug-resistant gene OXA-23 and a drug-resistant probe matched with the drug-resistant primer pair; The upstream primer sequence of the specific primer pair is shown in SEQ ID NO: 1, the downstream primer sequence of the primer of the specific primer pair is shown in SEQ ID NO: 2, and the specific probe sequence is shown in SEQ ID NO: 3; The upstream primer sequence of the drug-resistant primer pair is shown in SEQ ID NO: 4, the downstream primer sequence of the drug-resistant primer pair is shown in SEQ ID NO: 5, and the drug-resistant probe sequence is shown in SEQ ID NO: 6; The steps include: S1. Extract DNA from the sample to be tested; S2. Using the extracted DNA as a template, a duplex qPCR reaction system with a total volume of 20 μL was constructed, and specific primer pairs and specific probes at a concentration of 10 μM as well as drug-resistant primer pairs and drug-resistant probes were added to perform qPCR amplification reactions; S3. Verify the detection limit and linear relationship of 16sRNA and OXA-23 in the dual qPCR reaction system.

7. The method for detecting carbapenem-resistant Acinetobacter baumannii according to claim 6, characterized in that: In step S2, the qPCR amplification step is: 39 cycles are performed in the order of 95°C pre-denaturation for 30 seconds - 95°C denaturation for 5 seconds - 60°C annealing for 30 seconds.

8. The method for detecting carbapenem-resistant Acinetobacter baumannii according to claim 6, characterized in that: The annealing temperature of the dual qPCR reaction system is 57.3°C-58.4°C.

Images