Primer and probe combination for detecting carbapenem drug-resistant genes and common CRO and application of primer and probe combination

Abstract

The invention discloses a primer and probe combination for detecting carbapenem drug resistance genes and common CRO, the primer and probe combination is required by LAMP amplification, and the carbapenem drug resistance genes are selected from a KPC gene, an NDM gene, an OXA-48 gene, a VIM gene and an IMP gene; the common CRO is selected from the group consisting of: KP, PA, and AB. The invention further discloses a kit and a detection method for detecting the carbapenem drug resistance genes and the common CRO. The primer and probe combination provided by the invention is good in specificity and high in sensitivity, can be used for detecting five common drug-resistant genes of the penicillinase and identifying KP, PA and AB of common carbapenemase, and provides a certain basis for clinical treatment of carbapenem drug-resistant enterobacter bacterial infection.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular detection, and in particular, relates to a primer and probe combination for detecting carbapenem resistance genes and common CROs and an application thereof. Background Art

[0002] Bacterial resistance has become a major challenge in the field of global public health, among which carbapenem-resistant Gram-negative bacteria, also known as carbapenem-resistant bacteria (CRO), are the most severe forms of infection. CRO mainly includes carbapenem-resistant Enterobacteriales (CRE), carbapenem-resistant Pseudomonas aeruginosa (CRPA) and carbapenem-resistant Acinetobacter baumannii (CRAB), and is one of the key pathogens monitored in clinical hospital infections. Carbapenem antibiotics are a class of β-lactamase antibiotics with a broad antibacterial spectrum and the strongest antibacterial activity. Because of their stability to β-lactamase and low toxicity, they have become one of the most important antibacterial drugs for the treatment of CRO. However, with the widespread use of carbapenem antibiotics, resistant strains continue to emerge, which are prone to recurrent infections, resulting in treatment failure and increased mortality. The main reason for the resistance of resistant strains is the production of carbapenemases, and it is imperative to carry out the detection of carbapenemases.

[0003] At present, there are two methods for laboratory detection of carbapenemases: phenotypic detection and genotypic detection. Phenotypic detection methods include Carba NP test, modified carbapenem inactivation test, and carbapenemase inhibitor enhancement test. Among them, CarbaNP test uses homemade reagents, and some reagents have a short validity period; modified carbapenem inactivation test and carbapenemase inhibitor enhancement test both require overnight culture. Genotypic detection methods include enzyme immunochromatography technology and gene detection technology, and enzyme immunochromatography technology is relatively expensive. Genotypic detection includes real-time fluorescence quantitative PCR, whole genome sequencing (WGS), etc. Real-time fluorescence quantitative PCR has high sensitivity and specificity but relies on precision instruments, and the detection cost is relatively high. Whole genome sequencing (WGS) can comprehensively analyze the spectrum of drug-resistant genes, but data analysis is complex and time-consuming, and requires high professionalism.

[0004] Therefore, the development of a rapid, sensitive, specific, easy-to-operate, and low-cost detection kit for carbapenem resistance genes and common CROs is of great significance in guiding clinicians to choose appropriate drugs, avoid aggravating bacterial resistance and delay clinical diagnosis and treatment. Summary of the invention

[0005] The purpose of the present invention is to make up for the deficiencies of the prior art and provide a primer and probe combination for detecting carbapenem resistance genes and common CRE, which has the advantages of multiple detection indicators, low cost, high specificity, high sensitivity, short reaction time, and easy operation.

[0006] In order to achieve the above object, the first aspect of the present invention provides a primer and probe combination for detecting carbapenem resistance genes and common CROs, wherein the primer and probe combination is required for LAMP amplification and comprises:

[0007] (1) outer primers shown in SEQ ID NOs. 1 to 2, inner primers shown in SEQ ID NOs. 3 to 4, loop primers shown in SEQ ID NOs. 5 to 6, and a probe shown in SEQ ID NO. 7;

[0008] (2) outer primers shown in SEQ ID NOs. 8 to 9, inner primers shown in SEQ ID NOs. 10 to 11, loop primers shown in SEQ ID NOs. 12 to 13, and a probe shown in SEQ ID NO. 14;

[0009] (3) outer primers shown in SEQ ID NOs. 15 to 16, inner primers shown in SEQ ID NOs. 17 to 18, loop primers shown in SEQ ID NOs. 19 to 20, and a probe shown in SEQ ID NO. 21;

[0010] (4) outer primers shown in SEQ ID NOs. 22 to 23, inner primers shown in SEQ ID NOs. 24 to 25, loop primers shown in SEQ ID NOs. 26 to 27, and a probe shown in SEQ ID NO. 28;

[0011] (5) outer primers shown in SEQ ID NOs. 29 to 30, inner primers shown in SEQ ID NOs. 31 to 32, loop primers shown in SEQ ID NOs. 33 to 34, and a probe shown in SEQ ID NO. 35;

[0012] (6) outer primers shown in SEQ ID NOs. 36 to 37, inner primers shown in SEQ ID NOs. 38 to 39, loop primers shown in SEQ ID NOs. 40 to 41, and a probe shown in SEQ ID NO. 42;

[0013] (7) outer primers shown in SEQ ID NOs. 43 to 44, inner primers shown in SEQ ID NOs. 45 to 46, loop primers shown in SEQ ID NOs. 47 to 48, and a probe shown in SEQ ID NO. 49; and

[0014] (8) Outer primers represented by SEQ ID NOs. 50 to 51, inner primers represented by SEQ ID NOs. 52 to 53, loop primers represented by SEQ ID NOs. 54 to 55, and a probe represented by SEQ ID NO. 56.

[0015] In the present invention, the carbapenem resistance gene is selected from: Klebsiella pneumoniae carbapenemase KPC gene, New Delhi metallo-β-lactamase NDM gene, oxacillinase OXA-48 gene, Verona integron-encoded metallo-lactamase VIM gene, and imipenemase IMP gene; the common CRO is selected from: Klebsiella pneumoniae KP, Pseudomonas aeruginosa PA, and Acinetobacter baumannii AB.

[0016] Furthermore, the primers and probe of (1) are used to detect the KPC gene, the primers and probe of (2) are used to detect the NDM gene, the primers and probe of (3) are used to detect the OXA-48 gene, the primers and probe of (4) are used to detect the VIM gene, the primers and probe of (5) are used to detect the IMP gene, the primers and probe of (6) are used to detect KP, the primers and probe of (7) are used to detect PA, and the primers and probe of (8) are used to detect AB.

[0017] According to a preferred embodiment of the present invention, the target probes shown in SEQ ID NO.7, SEQ ID NO.14, SEQ ID NO.21, SEQ ID NO.28, SEQ ID NO.35, SEQ ID NO.42, SEQ ID NO.49, and SEQ ID NO.56 are modified with a FAM fluorescent group on the base T at the 5′ end or near the 5′ end, the 3′ end is modified with a BHQ1 quenching group, and the 3′ end The third or fourth base is modified to an RNA base.

[0018] In a second aspect of the present invention, a kit for detecting carbapenem resistance genes and common CROs is provided, wherein the kit comprises the primer and probe combination as described above.

[0019] Furthermore, the kit also includes outer primers shown in SEQ ID NOs. 57 to 58, inner primers shown in SEQ ID NOs. 59 to 60, loop primers shown in SEQ ID NOs. 61 to 62, and a probe shown in SEQ ID NO. 63 for detecting an internal standard gene.

[0020] According to the present invention, the internal standard gene is the GAPDH gene.

[0021] Preferably, the internal standard probe shown in SEQ ID NO.63 is modified with a ROX fluorescent group at the 5′ end, a BHQ2 quenching group at the 3′ end, and the third base at the 3′ end is modified to an RNA base.

[0022] The third aspect of the present invention provides a method for detecting carbapenem resistance genes and common CROs, the method comprising using the primer and probe combination or the kit as described above to perform a LAMP amplification reaction on a sample to be tested, and determining whether the sample to be tested contains carbapenem resistance genes or common CROs based on the amplification results.

[0023] According to a preferred embodiment of the present invention, the LAMP amplification reaction is a dual real-time fluorescent LAMP, including using the LAMP primer and probe combination as described above to amplify the sample DNA to be tested. If the corresponding target produces an S-shaped amplification curve, the target is positive, and if there is no S-shaped amplification curve, it is negative.

[0024] The primer and probe combination provided by the present invention has good specificity and high sensitivity, and can be used for the detection of five common carbapenemase resistance genes (KPC, NDM, OXA48, VIM, IMP) and the identification of common carbapenemase-producing Klebsiella pneumoniae KP, Pseudomonas aeruginosa PA, and Acinetobacter baumannii AB, providing a certain basis for the clinical treatment of carbapenem-resistant Enterobacteriales bacterial infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The steps of detecting a sample using the kit in Example 2 are shown.

[0026] Figure 2 The sensitivity test results of Example 4 are shown, wherein: A corresponds to KPC, B corresponds to NDM, C corresponds to OXA48, D corresponds to VIM, E corresponds to IMP, F corresponds to KP, G corresponds to PA, and H corresponds to AB.

[0027] Figure 3-1 and Figure 3-2 These are the LAMP and NGS sequencing results of 48 clinical sputum samples and 26 bronchoalveolar lavage fluid samples in Example 5. DETAILED DESCRIPTION

[0028] The present invention is further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0029] The experimental methods in the following examples, unless otherwise specified, are conventional methods and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0030] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0031] Example 1: Primer and probe screening

[0032] The present invention selects five carbapenem resistance genes, including Klebsiella pneumoniae carbapenemase (KPC), New Delhi metallo-β-lactamase (NDM), oxacillinase (OXA-48), metallo-lactamase encoded by Verona integron (VIM) and imipenemase (IMP), and three common carbapenemase-producing bacteria, including Klebsiella pneumoniae (KP), Pseudomonas aeruginosa (PA) and Acinetobacter baumannii (AB), as detection targets. Five sets of primers and probes were designed for each target, and a set of primer-probe combinations with good sensitivity and high specificity were screened out through plasmid gradient detection, as shown in Table 1 below.

[0033] Table 1: Primer probe sequences

[0034]

[0035]

[0036]

[0037] The target probes of the five genes (KPC, NDM, OXA48, VIM, IMP) and three common CROs (KP, PA, AB) are shown as SEQ ID NO.7, SEQ ID NO.14, SEQ ID NO.21, SEQ ID NO.28, SEQ ID NO.35, SEQ ID NO.42, SEQ ID NO.49, and SEQ ID NO.56, respectively, and the base T at the 5′ end or near the 5′ end thereof is modified with a FAM fluorescent group, the 3′ end is modified with a BHQ1 quenching group, and the 3′ third or fourth base at the 3′ end is modified to an RNA base; the internal standard probe is shown as SEQ ID NO.63, and the 5′ end thereof is modified with a ROX fluorescent group, the 3′ end is modified with a BHQ2 quenching group, and the 3′ third base at the 3′ end is modified to an RNA base.

[0038] Example 2, kit and detection method

[0039] This embodiment provides a kit for detecting carbapenem resistance genes and common CROs, which is composed of the following components:

[0040] (1) a detection reaction solution prepared by mixing the primer probes of each target shown in Table 1 of Example 1 with the primer probe of the internal standard GAPDH and LAMP freeze-dried beads (Wuhan Hanhai New Enzyme Biotechnology Co., Ltd., HMD5219-01, 25 μL reaction system, containing Bst enzyme and its buffer); (2) a nucleic acid extraction or purification reagent (Hangzhou Haiji Biotechnology Co., Ltd.); (3) a negative control (ddH2O); and (4) a positive control (containing 10 μL of the gene fragment of each target). 5 copies / μL of plasmid).

[0041] The test reaction solution formula (Formula 1) for KPC, NDM, IMP, PA and AB is shown in the following Table 2. For simplicity, "XX" in the table represents each carbapenem resistance gene or CRO.

[0042] Table 2: Recipe 1

[0043]

[0044]

[0045] The test reaction solution formula (Formula 2) for OXA48, VIM and KP is shown in the following Table 3. For simplicity, "XX" in the table represents each carbapenem resistance gene or CRO.

[0046] Table 3: Recipe 2

[0047] Components Volume (μL) Final concentration (μM) XX-F3(10μM) 0.6 0.24 XX-B3(10μM) 0.6 0.24 XX-FIP (10 μM) 4.8 1.92 XX-BIP (10 μM) 4.8 1.92 XX-LF(10μM) 1.2 0.48 XX-LB (10 μM) 1.2 0.48 XX-P1(10μM) 1.8 0.36 GAPDH-F3 (10 μM) 0.15 0.06 GAPDH-B3 (10 μM) 0.15 0.06 GAPDH-FIP (10 μM) 1.2 0.48 GAPDH-BIP (10 μM) 1.2 0.48 GAPDH-LF (10 μM) 0.3 0.12 GAPDH-LB (10 μM) 0.2 0.12 GAPDH-LF-P1 (10 μM) 0.45 0.09 LAMP freeze-dried pellets 1 piece 1× <![CDATA[ddH2O]]> 1.35 / Total 20 /

[0048] The steps of using the above kit to detect samples are as follows Figure 1 As shown, the details are as follows:

[0049] S1. Sample extraction:

[0050] Sputum: Use a pipette to draw 400 μL of nucleic acid extraction or purification reagent into a 1.5 mL centrifuge tube, then draw 100 μL of sputum into the centrifuge tube and oscillate to mix for 30 seconds; place in a 95°C metal bath for 10 minutes, inverting and mixing twice; then centrifuge at 7000 rpm for 2 minutes.

[0051] Alveolar lavage fluid: Use a pipette to draw 400 μL of nucleic acid extraction or purification reagent and add it to a 1.5 mL centrifuge tube, then draw 200 μL of alveolar lavage fluid and add it to the centrifuge tube, oscillate and mix for 30 seconds; place it in a 95°C metal bath for 10 minutes, inverting and mixing twice during this period; then centrifuge at 7000 rpm for 2 minutes.

[0052] S2. Sample addition: Use a pipette to add 5 μL of negative control, test sample nucleic acid and positive control into the corresponding reagent reaction tubes, cover the tubes tightly, vortex to mix, and centrifuge for 5 seconds.

[0053] S3. Hole layout

[0054]

[0055] S4. On-machine testing (nucleic acid amplification area)

[0056] Place the reaction tubes with samples in the constant temperature amplification instrument in sequence, mark them, and amplify according to the following procedure:

[0057] Reaction procedure: Bla-Lamp-Prboe: 65°C, 25 min.

[0058] S5. Interpretation of results (interpretation of each well individually)

[0059]

[0060] Note: There are two ways to judge the results: qualitative and quantitative. Qualitative means to directly judge the positive and negative according to the indicator light; quantitative means to judge the TTR value according to the amplification curve when the indicator light shows positive. If the corresponding target produces an S-type amplification curve, the target is positive, and if there is no S-type amplification curve, it is negative.

[0061] Example 3: Specificity Detection

[0062] In order to verify the specificity of the primer and probe combination of the present invention, the national reference product of the carbapenem resistance gene nucleic acid detection kit purchased from the China Food and Drug Identification Institute was reconstituted with 1 mL of nuclease-free sterile water and tested according to the method of Example 2. The results are shown in Table 4 below.

[0063] Table 4: Specificity test results

[0064]

[0065] From the results in Table 4, it can be seen that the specificity of each target (KPC, NDM, OXA48, VIM, IMP, KP, PA, AB) is good, indicating that the specificity of the primer and probe combination of the present invention is good.

[0066] Example 4: Sensitivity Detection

[0067] The concentration of the internal standard plasmid was 10 5 The plasmids of eight targets, KPC, NDM, OXA48, VIM, IMP, KP, PA and AB, were diluted to different concentrations (50, 10, 10, 20, 30, 40, 50, 60, 70, 80, 70, 80, 80, 90, 100, 100, 200, 300, 400, 500, 2 , 10 3 , 10 4 , 10 5 copies / μL), and each concentration gradient sample was tested according to the method of Example 2. The well position arrangement is shown in Table 5 below.

[0068] Table 5: Vacancy arrangement

[0069] Hole Position A B C D E F G H template <![CDATA[10 5 ]]> <![CDATA[10 4 ]]> <![CDATA[10 3 ]]> <![CDATA[10 2 ]]> 50 50 NTC NTC

[0070] Test results such as Figure 2 As shown in AH, each target plasmid is 5In the concentration range of ~50 copies / μL, both the internal standard and the target are amplified and the amplification curves show an obvious "S" shape, and the instrument displays a "yellow light"; neither the negative control NTC internal standard nor the target is amplified, and the instrument displays a "purple light".

[0071] The above test results prove that when the primer and probe combination of the present invention is used for detection, the detection sensitivity of each target (KPC, NDM, OXA48, VIM, IMP, KP, PA, AB) reaches 50 copies / μL.

[0072] Example 5: Clinical sample testing

[0073] 48 sputum samples and 26 bronchoalveolar lavage fluid samples from the clinic were tested using the method of Example 2; at the same time, all samples were subjected to LAMP and NGS sequencing simultaneously. The results are shown in Figure 3-1 and Figure 3-2 .

[0074] Taking the NGS results as the standard, the statistical results are shown in Table 6 below.

[0075] Table 6: Clinical sample test results

[0076] KPC NDM OXA48 VIM IMP KP PA AB Sensitivity 28 / 28 23 / 23 15 / 16 5 / 5 7 / 7 23 / 25 17 / 17 22 / 22 Specificity 46 / 46 51 / 51 58 / 58 69 / 69 67 / 67 49 / 49 57 / 57 52 / 52

[0077] The results of Table 6 are converted into percentages, as shown in Table 7.

[0078] Table 7: Clinical sample test results (percentage)

[0079] KPC NDM OXA48 VIM IMP KP PA AB Sensitivity 100％ 100％ 94％ 100％ 100％ 92％ 100％ 100％ Specificity 100％ 100％ 100％ 100％ 100％ 100％ 100％ 100％

[0080] Taking the NGS results as the standard, the primer and probe combination of the present invention was used for detection, and the specificity of each target (KPC, NDM, OXA48, VIM, IMP, KP, PA, AB) was 100%, and the sensitivity was above 90%.

[0081] The above description is only a partial embodiment of the present invention and is not intended to limit the scope of the present invention. The above embodiments of the present invention can also be modified in various ways. All simple and equivalent changes and modifications made according to the contents of the specification of the present invention fall within the protection scope of the present invention. The contents not described in detail in the present invention are all conventional technical contents.

Claims

1. A primer and probe combination for detecting carbapenem resistance genes and common CROs, characterized in that: The primer and probe combination is required for LAMP amplification, including: (1) outer primers shown in SEQ ID NOs. 1 to 2, inner primers shown in SEQ ID NOs. 3 to 4, loop primers shown in SEQ ID NOs. 5 to 6, and a probe shown in SEQ ID NO. 7; (2) outer primers shown in SEQ ID NOs. 8 to 9, inner primers shown in SEQ ID NOs. 10 to 11, loop primers shown in SEQ ID NOs. 12 to 13, and a probe shown in SEQ ID NO. 14; (3) outer primers shown in SEQ ID NOs. 15 to 16, inner primers shown in SEQ ID NOs. 17 to 18, loop primers shown in SEQ ID NOs. 19 to 20, and a probe shown in SEQ ID NO. 21; (4) outer primers shown in SEQ ID NOs. 22 to 23, inner primers shown in SEQ ID NOs. 24 to 25, loop primers shown in SEQ ID NOs. 26 to 27, and a probe shown in SEQ ID NO. 28; (5) outer primers shown in SEQ ID NOs. 29 to 30, inner primers shown in SEQ ID NOs. 31 to 32, loop primers shown in SEQ ID NOs. 33 to 34, and a probe shown in SEQ ID NO. 35; (6) outer primers shown in SEQ ID NOs. 36 to 37, inner primers shown in SEQ ID NOs. 38 to 39, loop primers shown in SEQ ID NOs. 40 to 41, and a probe shown in SEQ ID NO. 42; (7) outer primers shown in SEQ ID NOs. 43 to 44, inner primers shown in SEQ ID NOs. 45 to 46, loop primers shown in SEQ ID NOs. 47 to 48, and a probe shown in SEQ ID NO. 49; and (8) Outer primers represented by SEQ ID NOs. 50 to 51, inner primers represented by SEQ ID NOs. 52 to 53, loop primers represented by SEQ ID NOs. 54 to 55, and a probe represented by SEQ ID NO.

56.

2. The primer and probe combination according to claim 1, characterized in that: The carbapenem resistance gene is selected from: Klebsiella pneumoniae carbapenemase KPC gene, New Delhi metallo-β-lactamase NDM gene, oxacillinase OXA-48 gene, Verona integron-encoded metallo-lactamase VIM gene, and imipenemase IMP gene; the common CRO is selected from: Klebsiella pneumoniae KP, Pseudomonas aeruginosa PA, and Acinetobacter baumannii AB.

3. The primer and probe combination according to claim 2, characterized in that: The primers and probe of (1) are used to detect the KPC gene, the primers and probe of (2) are used to detect the NDM gene, the primers and probe of (3) are used to detect the OXA-48 gene, the primers and probe of (4) are used to detect the VIM gene, the primers and probe of (5) are used to detect the IMP gene, the primers and probe of (6) are used to detect KP, the primers and probe of (7) are used to detect PA, and the primers and probe of (8) are used to detect AB.

4. The primer and probe combination according to claim 1, characterized in that: The target probes shown in SEQ ID NO.7, SEQ ID NO.14, SEQ ID NO.21, SEQ ID NO.28, SEQ ID NO.35, SEQ ID NO.42, SEQ ID NO.49, and SEQ ID NO.56 are modified with a FAM fluorescent group on the base T at the 5′ end or near the 5′ end, modified with a BHQ1 quenching group at the 3′ end, and the 3rd or 4th base at the 3′ end is modified to an RNA base.

5. A kit for detecting carbapenem resistance genes and common CROs, characterized in that: The kit comprises the primer and probe combination according to any one of claims 1 to 4.

6. The kit according to claim 5, characterized in that It also includes outer primers shown in SEQ ID NOs. 57 to 58, inner primers shown in SEQ ID NOs. 59 to 60, loop primers shown in SEQ ID NOs. 61 to 62, and a probe shown in SEQ ID NO. 63 for detecting an internal standard gene.

7. The kit according to claim 6, characterized in that The internal standard gene is the GAPDH gene.

8. The kit according to claim 6 or 7, characterized in that The internal standard probe shown in SEQ ID NO.63 is modified with a ROX fluorescent group at the 5′ end, a BHQ2 quenching group at the 3′ end, and the third base at the 3′ end is modified to an RNA base.

9. A method for detecting carbapenem resistance genes and common CROs, characterized in that: The method comprises using the primer and probe combination of any one of claims 1 to 4 or the kit of any one of claims 5 to 8 to perform a LAMP amplification reaction on the sample to be tested, and determining whether the sample to be tested contains a carbapenem resistance gene or a common CRO according to the amplification result.

10. The method according to claim 9, characterized in that The LAMP amplification reaction is a dual real-time fluorescent LAMP, which includes amplifying the sample DNA to be tested using the LAMP primer and probe combination of claim 1. If the corresponding target produces an S-shaped amplification curve, the target is positive, and if there is no S-shaped amplification curve, it is negative.

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