Composition for detecting acinetobacter baumannii and beta lactamase drug-resistant gene of acinetobacter baumannii

Abstract

The invention belongs to the field of molecular biological detection, and provides a joint detection composition which is mainly used for detecting acinetobacter baumannii and a drug-resistant gene thereof by utilizing a multiple fluorescent PCR (Polymerase Chain Reaction) analysis method, so that at least four targets in pgaD, NDM, OXA23, OXA24, OXA51 and OXA58 are simultaneously detected and distinguished in a single-tube reaction system. Meanwhile, the detection sensitivity of the composition is higher and reaches 500 copies / mL, and the detection is more accurate.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biological detection, and specifically relates to a composition for detecting and distinguishing targets such as pgaD, NDM, OXA23, OXA24, OXA51, and OXA58 for detecting Acinetobacter baumannii and its beta-lactamase resistance. Background Art

[0002] Acinetobacter baumannii was considered a low-virulence bacterium until the mid-1990s, when its severity was not taken seriously. In recent years, public health agencies have paid more and more attention to its increasing virulence and the difficulty in treating infections caused by drug resistance. Acinetobacter baumannii is a Gram-negative bacterium that is a strictly aerobic, catalase-positive, oxidase-negative, and non-lactose-fermenting opportunistic pathogen. Acinetobacter baumannii is found almost everywhere, such as water bodies, soil, mines, crude oil, sewage, sludge, solid surfaces, human skin, and wildlife. It is not only difficult to treat, but also difficult to eliminate. This is due to its resistance to desiccation, seasonal adaptability, and high temperature tolerance, as well as reduced sensitivity to disinfectants and biofilm protection. Drug-resistant Acinetobacter baumannii is one of the most common pathogens of nosocomial infections, especially in immunocompromised patients and ICU wards. In addition, long-term use of antibiotics, major surgery, severe burns, and immunosuppression increase the risk of Acinetobacter baumannii infection. Acinetobacter baumannii infection can lead to ventilator-associated pneumonia, bacteremia, urinary tract infection, and meningitis. In 2019, more than 600,000 deaths were associated with bacterial resistance, of which 145,000 deaths were attributable to bacterial resistance. The number one resistant bacteria attributable to deaths was carbapenem-resistant Acinetobacter baumannii (18,143 cases). Meanwhile, in 2019, the World Health Organization stated that Acinetobacter baumannii is considered the most dangerous multidrug-resistant bacteria. The overall prevalence of multidrug-resistant strains of Acinetobacter baumannii in patients with hospital-acquired pneumonia and ventilator-associated pneumonia is estimated to be 79.9%, and the overall mortality rate can be as high as 56.2%.

[0003] In view of this, it is particularly important to clarify its drug resistance. Therefore, this field requires a product that can simply and quickly detect the drug resistance of Acinetobacter baumannii with high sensitivity and good specificity, which is conducive to providing timely clinical treatment strategies. Summary of the invention

[0004] In view of this, in a first aspect, the present invention provides a composition for detecting Acinetobacter baumannii and its β-lactamase resistance gene, comprising upstream and downstream primers and probes for detecting at least 4 of the following targets:

[0005] Upstream and downstream primers and probes for detecting pgaD as shown in SEQ ID NOs: 1 to 3;

[0006] Upstream and downstream primers and probes for detecting NDM as shown in SEQ ID NOs: 4 to 6;

[0007] Upstream and downstream primers and probes for detecting OXA23 as shown in SEQ ID NOs: 7 to 9;

[0008] Upstream and downstream primers and probes for detecting OXA24 as shown in SEQ ID NOs: 10 to 12;

[0009] Upstream and downstream primers and probes for detecting OXA51 as shown in SEQ ID NOs: 13 to 15; or

[0010] The upstream and downstream primers and probes for detecting OXA58 are shown in SEQ ID NOs: 16-18.

[0011] The joint detection composition provided by the present invention mainly uses a multiplex fluorescence PCR analysis method to detect Acinetobacter baumannii and its β-lactamase resistance gene, thereby simultaneously realizing the detection and differentiation of at least 4 targets of pgaD, NDM, OXA23, OXA24, OXA51, and OXA58 in a single-tube reaction system, so as to provide a targeted strategy for subsequent treatment. At the same time, the composition of the present invention has a higher detection sensitivity of 500 copies / mL, and the detection is more accurate, which is conducive to providing prevention policies in a timely manner.

[0012] The present invention provides a composition for detecting Acinetobacter baumannii and its β-lactamase resistance gene, comprising:

[0013] Upstream and downstream primers and probes for detecting pgaD as shown in SEQ ID NOs: 1 to 3;

[0014] Upstream and downstream primers and probes for detecting NDM as shown in SEQ ID NOs: 4 to 6;

[0015] Upstream and downstream primers and probes for detecting OXA23 as shown in SEQ ID NOs: 7 to 9;

[0016] Upstream and downstream primers and probes for detecting OXA24 as shown in SEQ ID NOs: 10 to 12;

[0017] Upstream and downstream primers and probes for detecting OXA51 as shown in SEQ ID NOs: 13 to 15; and

[0018] The upstream and downstream primers and probes for detecting OXA58 are shown in SEQ ID NOs: 16-18.

[0019] Furthermore, the composition includes upstream and downstream primers and probes for detecting the internal standard.

[0020] In some specific embodiments, the internal standard is a human internal standard gene.

[0021] In a specific embodiment, the internal standard is a human housekeeping gene.

[0022] Furthermore, the fluorescent groups of different probes in the composition of the present invention are different from each other and do not interfere with each other.

[0023] In this article, "different from each other and not interfering with each other" means that the fluorescent groups used in each probe in the composition are different and will not affect each other's detection, that is, different channels can be used for detection. For example, ATTO425, Quasar705, FAM, HEX, ROX, CY5 and CY5.5 can be used. The absorbance values ​​of these groups are not close, and different channels can be selected, so they will not interfere with each other.

[0024] Further, in some embodiments, the composition of the present invention may include one or more of the above primer and probe sets. In the present invention, "set" refers to upstream primers, downstream primers and probes that match each other to detect a target.

[0025] The composition of the present invention can be combined into any combination of the corresponding six targets. Those skilled in the art can combine as needed to detect which targets, that is, to combine the primers and probe pairs corresponding to the targets. These combinations are all included in the present invention.

[0026] For example, it may include any 5 of the above 6 sets of primers and probes, it may include any 4 of the above 6 sets of primers and probes, it may include any 3 of the above 6 sets of primers and probes, it may include any 2 of the above 6 sets of primers and probes, or it may include any 1 of the above 6 sets of primers and probes.

[0027] In some specific embodiments, the compositions of the present invention are used for fluorescent PCR.

[0028] In a specific embodiment, the fluorescent reporter group of the probe of pgaD is FAM; the fluorescent reporter group of the probe of NDM is HEX (or VIC); the fluorescent reporter group of the probe of OXA23 is ATTO 425; the fluorescent reporter group of the probe of OXA24 is ROX; the fluorescent reporter group of the probe of OXA51 is CY5; and the fluorescent reporter group of the probe of OXA58 is Quasar705.

[0029] Furthermore, the 3' end of the probe also has a non-fluorescent quencher.

[0030] Furthermore, the 3' end of the probe also has a quencher group, such as MGB, Dabcyl BHQ-0, BHQ1, BHQ2 or BHQ3.

[0031] Furthermore, the dosage of a single primer in the composition is 0.01 to 0.5 μM; the dosage of a single probe in the composition is 0.005 to 0.2 μM.

[0032] In a specific embodiment, each component of the composition of the present invention is present in a separate package.

[0033] In a particular embodiment, the components of the composition of the present invention are present in the same package.

[0034] Furthermore, the components of the composition of the present invention are present in a mixed form.

[0035] In a second aspect, the present invention provides the use of the above-mentioned composition of the present invention in the preparation of a kit for detecting Acinetobacter baumannii and its β-lactamase resistance gene, wherein the target is at least 4 of pgaD, NDM, OXA23, OXA24, OXA51, and OXA58.

[0036] In a third aspect, the present invention provides a kit for detecting Acinetobacter baumannii and its β-lactamase resistance gene, wherein the kit comprises the composition of the present invention as described above.

[0037] Furthermore, the kit also includes a negative quality control product and a positive quality control product.

[0038] In a specific embodiment, the negative control is at least one of DEPC H2O, physiological saline, and an internal standard gene. The positive control is at least one of the plasmid or fragment genes of pgaD, NDM, OXA23, OXA24, OXA51, and OXA58.

[0039] Furthermore, the kit also includes a nucleic acid amplification reagent.

[0040] Furthermore, the amplification reagent includes dNTP(U)s, PCR buffer, DNA polymerase, and Mg 2+ At least one of .

[0041] Furthermore, the kit also includes: a nucleic acid releasing reagent and a nucleic acid extracting reagent.

[0042] Furthermore, the concentration of the DNA polymerase is 1 U / reaction to 15 U / reaction, for example, the DNA polymerase may be Taq enzyme.

[0043] In a specific embodiment, the kit of the present invention comprises: Taq enzyme, Mg 2+ , dNTP(U)s, primers, probes, UNG enzyme and PCR buffer.

[0044] PCR buffer is composed of Tris-HCl, MgCl2, KCl, Triton X-100, etc. The total volume in a single PCR reaction tube is generally 20 μl to 200 μl.

[0045] In a fourth aspect, a method for detecting Acinetobacter baumannii and its β-lactamase resistance gene is provided, the method comprising the following steps:

[0046] 1) Extracting nucleic acid from the sample to be tested;

[0047] 2) using the composition of the present invention or the kit of the present invention as described above to perform fluorescent quantitative PCR on the nucleic acid obtained in step 1);

[0048] 3) Obtain and analyze the results.

[0049] In the present invention, the sample used for detection may be tissue fluid, serum, blood, etc., but is not limited thereto.

[0050] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0051] UNG enzyme reaction, temperature is 50-65℃, time is 1-5 minutes, 1 cycle; Taq enzyme activation, temperature is 90-99℃, time is 1-5 minutes, 1 cycle; denaturation, temperature is 95℃, time is 5-30 seconds, annealing, temperature is 55℃-60℃, time is 10-60 seconds, 30-50 cycles, and fluorescence is collected.

[0052] In a specific embodiment, a method for detecting Acinetobacter baumannii and its β-lactamase resistance gene for non-diagnostic purposes is provided, the method comprising the following steps:

[0053] 1) Extracting nucleic acid from the sample to be tested;

[0054] 2) using the composition of the present invention or the kit of the present invention as described above to perform fluorescent quantitative PCR on the nucleic acid obtained in step 1);

[0055] 3) Obtain and analyze the results.

[0056] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0057] UNG enzyme reaction, temperature is 50-65℃, time is 1-5 minutes, 1 cycle; Taq enzyme activation, temperature is 90-99℃, time is 1-5 minutes, 1 cycle; denaturation, temperature is 95℃, time is 5-30 seconds, annealing, temperature is 55℃-60℃, time is 10-60 seconds, 30-50 cycles, and fluorescence is collected.

[0058] In a specific embodiment, a composition is provided for preparing a reagent for detecting Acinetobacter baumannii and its β-lactamase resistance gene, wherein the use detection comprises the following steps:

[0059] 1) Extracting nucleic acid from the sample to be tested;

[0060] 2) using the composition of the present invention or the kit of the present invention as described above to perform fluorescent quantitative PCR on the nucleic acid obtained in step 1);

[0061] 3) Obtain and analyze the results.

[0062] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0063] UNG enzyme reaction, temperature is 50-65℃, time is 1-5 minutes, 1 cycle; Taq enzyme activation, temperature is 90-99℃, time is 1-5 minutes, 1 cycle; denaturation, temperature is 95℃, time is 5-30 seconds, annealing, temperature is 55℃-60℃, time is 10-60 seconds, 30-50 cycles, and fluorescence is collected. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figures 1 to 3 A graph showing the test results of different sample combinations of the composition of the present invention;

[0065] Figures 4 to 9 The sensitivity results of the composition of the present invention are shown in Figure 1 (pgaD, NDM, OXA23, OXA24, OXA51, and OXA58, respectively);

[0066] Fig.10 It is a specific result diagram of the composition of the present invention;

[0067] Figures 11 to 16 The anti-interference result diagram of the composition of the present invention (respectively pgaD, NDM, OXA23, OXA24, OXA51, OXA58);

[0068] Fig.17 It is a test result diagram of the comparative example composition of the present invention. DETAILED DESCRIPTION

[0069] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are used to illustrate the present invention, rather than to limit the present invention.

[0070] Example 1: Primers and probes used in the present invention

[0071] Table 1

[0072]

[0073]

[0074] The fluorescent reporter group of the probe of pgaD is FAM; the fluorescent reporter group of the probe of NDM is HEX (or VIC); the fluorescent reporter group of the probe of OXA23 is ATTO 425; the fluorescent reporter group of the probe of OXA24 is ROX; the fluorescent reporter group of the probe of OXA51 is CY5; and the fluorescent reporter group of the probe of OXA58 is Quasar 705.

[0075] Example 2: Method for detecting Acinetobacter baumannii and its β-lactamase resistance gene

[0076] 1. Reagent Preparation

[0077] 1.1 Take out the components in the box and place them at room temperature. After the temperature is balanced to room temperature, mix them well and centrifuge them for standby use.

[0078] 1.2 According to the number of samples to be tested, negative controls, and positive controls, take the corresponding amount of components in proportion (PCR reaction solution 44μL / person + enzyme mixture 1μL / person), mix them thoroughly to form a PCR mixture, and centrifuge it for standby use.

[0079] 1.3 Transfer the prepared reagents to the sample processing area for later use.

[0080] 2. Sample processing and loading

[0081] The sample tested in the present invention is sputum. The viral nucleic acid is extracted by magnetic bead method, and the following operations are performed in the sample processing room:

[0082] 2.1 Take a 1.5mL cryotube and add glass beads. Add 300μL of the treated sample and 300μL of TE to the 1.5mL cryotube containing the mixed glass beads and grind using a grinder. After grinding, take out the cryotube and centrifuge it instantly. Take out the supernatant liquid as the sample to be tested for later use.

[0083] 2.2 Take a number of 1.5mL centrifuge tubes according to the number of samples to be tested, and add 300μL of the sample to be tested to each tube;

[0084] 2.3 Add 500 μL of extraction solution 1 and 50 μL of proteinase K-magnetic bead mixture; cover the tube, shake and mix for 30 seconds, and heat at 60℃ for 10 minutes.

[0085] 2.4 Let stand at room temperature for 1 minute, centrifuge at low speed, place the centrifuge tube on the magnetic separator, and slowly discard the waste liquid after 5 minutes (*Be careful not to touch the magnetic beads adsorbed on the inner side of the tube wall);

[0086] 2.5 Add 200 μL of washing solution 1 and 600 μL of washing solution 2, shake and mix for 30 seconds, centrifuge at low speed, and place the centrifuge tube in a magnetic separator. Magnetic suction for 3 minutes, completely aspirate and discard the liquid.

[0087] 2.6 Place the centrifuge tube in a centrifuge and centrifuge it at a low speed for a short time. Place the centrifuge tube in a magnetic separator again and magnetically absorb it for 3 minutes to completely absorb the liquid at the bottom of the tube.

[0088] 2.7 Add 30-100 μL of elution buffer S (80 μL is recommended), shake and mix for 30 seconds to elute the magnetic beads on the wall of the centrifuge tube to the bottom of the tube, and let it stand at room temperature for 3 minutes; centrifuge at a low speed, place the centrifuge tube on the magnetic separator again for 3 minutes, and then transfer the eluted nucleic acid to a clean 1.5 mL centrifuge tube.

[0089] 2.8 Pipette 5 μL of the above-treated samples, positive controls and negative controls and add them to the corresponding 0.2 mL PCR reaction tubes, add 45 μL of PCR mixture to each tube and cover the tube.

[0090] Table 2

[0091]

[0092]

[0093] PCR amplification:

[0094] On a PCR instrument such as the SLAN-96P fully automatic medical PCR analysis system, PCR amplification is performed according to a certain temperature and time setting program. The PCR amplification program is set as shown in Table 3 below:

[0095] Table 3

[0096]

[0097] The specific test results are shown in Table 4.

[0098] Table 4. Interpretation of test results

[0099]

[0100] Note: If Acinetobacter baumannii is judged negative, but any resistance site is judged positive, it is another strain containing the resistance gene.

[0101] Example 3: Test results of the test samples of the composition of the present invention

[0102] The primers and probes shown in Example 1 were used to verify samples of pgaD, NDM, OXA23, OXA24, OXA51, and OXA58 according to the method of Example 2. The results showed that the composition of the present invention can detect and distinguish any combination of the above-mentioned drug-resistant genes. For example, the composition of the present invention detects:

[0103] pgaD, NDM, OXA51, OXA23 ( Figure 1 );

[0104] pgaD, NDM, OXA24, OXA23, OXA58( Figure 2 );

[0105] pgaD, NDM, OXA23, OXA24, OXA51, OXA58( Figure 3 ).

[0106] These figures prove that the present invention is a 6-plex PCR that can amplify the above 6 targets at one time and can be distinguished using a 6-channel PCR instrument. It also proves that any corresponding pair of combinations of the composition of the present invention can detect any corresponding combination of drug-resistant gene targets.

[0107] Example 4: Sensitivity of the composition of the present invention

[0108] Using the composition in Example 1 of the present invention, a gradient dilution of the plasmid was taken, and the concentrations were 500000, 50000, 5000, 500, 500, and 50 copies / ml, respectively. Five concentrations were tested, and 5 μL was taken as a template to simulate clinical samples and perform multiple PCR detection on a fluorescent quantitative PCR instrument. The results are as follows Figures 4 to 9 As shown, it is shown that each channel can still accurately detect samples as low as 500 copies / mL, indicating that the sensitivity of the composition of the present invention is 500 copies / mL.

[0109] Example 5. Specificity of the composition of the present invention

[0110] The composition of the present invention has no cross reaction with common pathogens and other pathogens with similar infection symptoms (Streptococcus pneumoniae, Haemophilus influenzae, Legionella pneumophila, Bordetella pertussis, Staphylococcus aureus, Mycoplasma pneumoniae, Chlamydia pneumoniae, Mycobacterium tuberculosis, nontuberculous mycobacteria, Rhodococcus equi, Candida albicans, Candida tropicalis, Candida krusei, influenza A virus, influenza B virus, respiratory syncytial virus). The results are as follows Fig.10 As shown, it is shown that the composition of the present invention has good specificity.

[0111] Example 6: Anti-interference performance of the composition of the present invention

[0112] The composition of the example was analyzed for interferences by conventional methods. The experimental results showed that potential PCR inhibitors / interfering substances such as 10 μg / mL mucin, 20 μg / mL heme, 20 μg / mL trimethoprim, 0.44 mg / mL sulfamethoxazole, 0.8 mg / mL amphotericin B, 0.1 g / mL itraconazole, 0.5 mg / mL fluconazole, 0.1 g / ml azithromycin, 1 mg / ml epinephrine, 4 mg / mL lidocaine hydrochloride, etc. at certain concentrations had no significant effect on the kit. The amplification results of the PCR reaction solution in the presence of infectious substances are shown below. Figures 11 to 16 shown.

[0113] Comparative Example 1: Other primers and probes designed by the present invention that have poor effects

[0114] Due to the principle of complementary base pairing, dimers will form between primers and (or) probes, but this probability is very small and can be ruled out at the beginning of the design. However, when multiple targets are detected together, there are many primers and probes, and dimers are likely to occur between primers and primers, probes and probes, or primers and probes. To ensure the conservatism of the design (conservatism is crucial to the accuracy of the detection), and to consider the mutual interference between different primers and probes, the primers and probes need to be carefully designed.

[0115] Therefore, the inventors also designed some other primers and probes to form a different detection system 2 (sequence not shown), which is also used to detect the above target gene. Fig.17 As shown in the figure, it can be seen that the detection performance of the six target genes is reduced, the Ct value is delayed, and the fluorescence increment is reduced.

Claims

1. A composition for detecting Acinetobacter baumannii and its β-lactamase resistance gene, comprising upstream and downstream primers and probes for detecting at least 4 of the following targets: Upstream and downstream primers and probes for detecting pgaD as shown in SEQ ID NOs: 1 to 3; Upstream and downstream primers and probes for detecting NDM as shown in SEQ ID NOs: 4 to 6; Upstream and downstream primers and probes for detecting OXA23 as shown in SEQ ID NOs: 7 to 9; Upstream and downstream primers and probes for detecting OXA24 as shown in SEQ ID NOs: 10 to 12; Upstream and downstream primers and probes for detecting OXA51 as shown in SEQ ID NOs: 13 to 15; or The upstream and downstream primers and probes for detecting OXA58 are shown in SEQ ID NOs: 16-18.

2. The composition according to claim 1, characterized in that The composition comprises: Upstream and downstream primers and probes for detecting pgaD as shown in SEQ ID NOs: 1 to 3; Upstream and downstream primers and probes for detecting NDM as shown in SEQ ID NOs: 4 to 6; Upstream and downstream primers and probes for detecting OXA23 as shown in SEQ ID NOs: 7 to 9; Upstream and downstream primers and probes for detecting OXA24 as shown in SEQ ID NOs: 10 to 12; Upstream and downstream primers and probes for detecting OXA51 as shown in SEQ ID NOs: 13 to 16; and The upstream and downstream primers and probes for detecting OXA58 are shown in SEQ ID NOs: 16-18.

3. The composition according to claim 1, characterized in that The composition also includes upstream and downstream primers and probes for detecting the internal standard.

4. The composition according to claim 1, characterized in that The fluorescent groups of different probes in the composition are different from each other and do not interfere with each other.

5. The composition according to claim 4, characterized in that The fluorescent reporter group of the probe of pgaD is FAM; the fluorescent reporter group of the probe of NDM is HEX (or VIC); the fluorescent reporter group of the probe of OXA23 is ATTO 425; the fluorescent reporter group of the probe of OXA24 is ROX; the fluorescent reporter group of the probe of OXA51 is CY5; and the fluorescent reporter group of the probe of OXA58 is Quasar 705.

6. The composition according to any one of claims 1 to 5, characterized in that The components of the composition are present in admixture.

7. Use of the composition according to any one of claims 1 to 6 in the preparation of a kit for detecting Acinetobacter baumannii and its β-lactamase resistance gene, wherein: The targets are at least four of pgaD, NDM, OXA23, OXA24, OXA51 or OXA58.

8. A kit for detecting Acinetobacter baumannii and its β-lactamase resistance gene, the kit comprising the composition according to any one of claims 1 to 6.

9. The kit according to claim 8, characterized in that The kit also includes at least one of a nucleic acid releasing reagent, a nucleic acid extracting reagent, and a nucleic acid amplifying reagent.

10. Use of a composition for preparing a reagent for detecting Acinetobacter baumannii and its β-lactamase resistance gene, the detection comprising the following steps: 1) Extracting nucleic acid from the sample to be tested; 2) performing fluorescent quantitative PCR analysis on the nucleic acid obtained in step 1) using the composition according to any one of claims 1 to 6 or the kit according to claim 8 or 9; 3) Obtain and analyze the results.

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