Primer probe combination and kit for real-time fluorescence MIRA detection of high-toxicity klebsiella pneumoniae and application of primer probe combination and kit

Abstract

The invention relates to the technical field of biology, in particular to a primer probe combination and a kit for real-time fluorescence MIRA detection of high-toxicity klebsiella pneumoniae and application of the primer probe combination and the kit. The invention provides a primer probe combination for detecting high-virulence klebsiella pneumoniae and matched products (a reagent and an MIRA detection kit). Aiming at the typical virulence gene iucA of the high-virulence klebsiella pneumoniae, the primer group and the probe suitable for real-time fluorescence MIRA detection are specifically designed, and the primer probe combination and the product are particularly suitable for on-site screening of the high-virulence klebsiella pneumoniae, are high in detection speed and good in specificity and are not interfered by false positive, cross contamination and the like. Meanwhile, the detection result is reliable, the sensitivity is high, the specificity is strong, and the kit is suitable for food inspection, disease control centers and quality supervision departments.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a primer-probe combination, a kit and applications thereof for real-time fluorescent MIRA detection of highly toxic Klebsiella pneumoniae. Background Art

[0002] Klebsiella pneumoniae is an important zoonosis pathogen widely found in the environment. It can be transmitted to humans through contamination of the food chain and water sources, causing serious community-acquired and hospital-acquired infections. In the 1980s, researchers first reported hypervirulent K. pneumoniae (hvKP). Compared with ordinary K. pneumoniae, hvKP has higher invasiveness and infection ability, and can cause more extensive and serious infections, such as liver abscess, necrotizing fasciitis and meningitis.

[0003] Traditionally, the identification of highly virulent Klebsiella pneumoniae is mainly through the string test and the detection of capsular antigen serotypes. However, these methods are not sensitive and specific enough, have high time costs, cannot accurately distinguish hvKP, and are highly operator-dependent. Therefore, in practice, there is an urgent need to find a simple, accurate and rapid identification method. With the advancement of genome sequencing and molecular biology, studies have found that genes such as iucA, peg-344, iroB, p-rmpA and p-rmpA2 can be used as accurate markers for identifying hvKP. The development of molecular biological rapid diagnostic technology based on these genes is of great practical significance for preventing and controlling the spread of hvKP.

[0004] Conventional nucleic acid detection methods such as conventional PCR sequencing are time-consuming and labor-intensive, require professional PCR equipment and experienced operators, and cannot meet the needs of rapid on-site detection. In recent years, a new isothermal amplification technology, Multienzyme Isothermal Rapid Amplification (MIRA), has attracted widespread attention from researchers due to its simplicity, rapidity, high sensitivity, and the lack of need for professional equipment. However, there is currently no rapid diagnosis method for hvKP based on real-time fluorescence quantitative MIRA (Real-time MIRA). Summary of the invention

[0005] The purpose of the present invention is to provide a primer-probe combination, a kit and its application for real-time fluorescent MIRA detection of highly virulent Klebsiella pneumoniae, so as to solve the problems existing in the above-mentioned prior art. The primer-probe combination provided by the present invention can specifically and rapidly detect highly virulent Klebsiella pneumoniae, and also has the advantage of high sensitivity.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a primer-probe combination for real-time fluorescent MIRA detection of highly virulent Klebsiella pneumoniae, wherein the primer-probe combination comprises an upstream primer having a nucleotide sequence as described in SEQ ID No.7, a downstream primer having a nucleotide sequence as described in SEQ ID No.8, and a probe (fluorescent probe) having a nucleotide sequence as described in SEQ ID No.9.

[0008] Further preferably, the probe is labeled with a FAM luminescent group at the 30th base T from the 5' end, the 31st base is replaced by tetrahydrofuran THF, the 32nd base T is labeled with a BHQ1 quenching group, and the 3' end is modified with a C3 spacer blocking.

[0009] Further preferably, the highly virulent Klebsiella pneumoniae is Klebsiella pneumoniae carrying the iucABCD gene cluster.

[0010] The present invention specifically designs primers and probes suitable for MIRA detection for the virulence gene iucA of highly toxic Klebsiella pneumoniae. The primer-probe combination and the product containing the primer-probe combination are particularly suitable for on-site screening of highly toxic Klebsiella pneumoniae, and has the advantages of fast detection speed, good specificity, no interference from false positives and cross contamination, reliable results, high sensitivity (detection limit is 1.68×10 0 pg / mL or 8×10 0 CFU / mL) and has the advantages of strong specificity. It can be seen that the primer-probe combination provided by the present invention can be used to prepare products for detecting and / or assisting in detecting highly virulent Klebsiella pneumoniae.

[0011] The present invention provides the use of the above primer-probe combination in the preparation of a product for detecting and / or assisting in the detection of highly virulent Klebsiella pneumoniae.

[0012] Further preferably, the application scenarios include the slaughtering line (slaughtering, disemboweling, rinsing, cutting), the production workshop (cleaning, mixing, sorting, heating, packaging), and various equipment, countertops, space environments and human medical environments involved in the storage, transportation and sales of meat, eggs and dairy products.

[0013] Further preferably, the human medical environment includes various medical and health systems; the various medical and health systems include diagnostic and treatment equipment and space environment of large, medium and small hospitals, clinics, community health service centers and health and epidemic prevention institutions.

[0014] Preferably, the product includes reagents, kits and chips.

[0015] Further preferably, the kit is a MIRA detection kit.

[0016] The present invention provides a product for detecting and / or assisting in detecting highly virulent Klebsiella pneumoniae, wherein the product comprises the above-mentioned primer-probe combination.

[0017] Preferably, the product also includes a rapid DNA extraction reagent, a reaction buffer and a freeze-dried enzyme powder.

[0018] Preferably, the rapid DNA extraction reagent comprises Chelex-100, lysozyme and Tris-EDTA buffer; the mass volume ratio of the Chelex-100, the lysozyme and the Tris-EDTA buffer is 5 g: 200 mg: 100 mL;

[0019] The reaction buffer contains 20 mM Tris-HCl, 15 mM (NH4)2SO4, 50 mM KCl, 5 mM MgSO4, 0.1 wt.% Tween-20, 1.5 mM dNTPs, 0.6 mg / mL BSA, 4 mM ATP, 30 mM creatine phosphate, 5.5% (w / v) polyethylene glycol 20000 and 2 mM dithiothreitol;

[0020] The freeze-dried enzyme powder includes 50 μg / mL Bst DNA polymerase, 10 μg / mL Nt.BstNBI endonuclease, 0.5 mg / mL single-stranded DNA binding protein, 500 ng / mL UvsX recombinase and 200 ng / μL creatine kinase.

[0021] Preferably, the product comprises a reagent and a kit.

[0022] Further preferably, the kit is a MIRA detection kit.

[0023] The present invention provides the use of the primer-probe combination or the product in detecting highly virulent Klebsiella pneumoniae for non-diagnostic and non-therapeutic purposes.

[0024] The present invention provides a method for detecting highly virulent Klebsiella pneumoniae for non-diagnostic and non-therapeutic purposes, comprising the following steps:

[0025] The DNA genome of the sample to be tested is used as the template DNA, the template DNA and the above-mentioned primer probe combination are mixed and reacted at a constant temperature, and real-time fluorescence MIRA detection is performed; if fluorescence appears, it is determined that the sample to be tested contains highly virulent Klebsiella pneumoniae.

[0026] Preferably, the isothermal reaction is carried out at a temperature of 37-42° C. and for a time of 15-20 min.

[0027] More preferably, the isothermal reaction is carried out at a temperature of 39° C. and for 20 min.

[0028] Further preferably, the samples to be tested are strains, swabs and milk, but are not limited to strains, swabs and milk.

[0029] Further preferably, during the real-time fluorescence MIRA detection process, the fluorescence signal is collected every 30 seconds (the selection of the signal collection channel is consistent with the probe design).

[0030] The present invention discloses the following technical effects:

[0031] The present invention specifically designs primers and probes suitable for MIRA detection for the virulence gene iucA of highly toxic Klebsiella pneumoniae. The primer-probe combination and the product containing the primer-probe combination are particularly suitable for on-site screening of highly toxic Klebsiella pneumoniae, and has the advantages of fast detection speed, good specificity, no interference from false positives and cross contamination, reliable results, high sensitivity (detection limit is 1.68×10 0 pg / mL or 8×10 0 CFU / mL) and has the advantages of strong specificity, and is suitable for use by food inspection, disease control centers, and quality supervision departments. It can be seen that the primer-probe combination for detecting highly virulent Klebsiella pneumoniae and the supporting products (reagents and MIRA detection kit) provided by the present invention can effectively detect highly virulent Klebsiella pneumoniae.

[0032] At the same time, through multi-enzyme constant temperature amplification and fluorescence signal detection, the specific MIRA primer probe combination provided by the present invention can quickly identify whether the sample contains highly virulent Klebsiella pneumoniae. Moreover, the detection method provided by the present invention has the advantages of being fast, low-cost, simple to operate, and not requiring professional equipment, and is suitable for rapid on-site detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0034] Figure 1 The fluorescence MIRA detection results of different primer sets;

[0035] Figure 2 The primer probe set designed for the present invention is for the specific detection of highly virulent Klebsiella pneumoniae;

[0036] Figure 3 The primer probe set designed for the present invention is sensitive for the detection of highly virulent Klebsiella pneumoniae;

[0037] Figure 4 The primer probe set designed for the present invention is used for accurate detection of highly virulent Klebsiella pneumoniae in actual samples;

[0038] Figure 5 The primer probe set designed for the present invention is used for sensitive detection of highly virulent Klebsiella pneumoniae in actual samples. DETAILED DESCRIPTION

[0039] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0040] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0041] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0042] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0043] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0044] Example 1 Design and screening of real-time MIRA specific primers and probes for detecting highly virulent Klebsiella pneumoniae

[0045] 1. Design of candidate specific primers and probes for highly virulent Klebsiella pneumoniae

[0046] Using the bioinformatics analysis tool BLAST, according to the Klebsiella pneumoniae nucleic acid sequence published by GenBank, the typical iucA gene of Klebsiella pneumoniae (GenBank: FO834905.1, whose CDS sequence is shown in SEQ ID No. 13, specifically:

[0047] >FO834905.1:58421-60145Klebsiella pneumoniae str.Kp52.145,plasmid II,complete genome

[0048]

[0049] Table 1 Candidate primer and probe sequences for the iucA gene

[0050]

[0051]

[0052] Note: F is the upstream primer, R is the downstream primer, and probe is the probe.

[0053] 2. MIRA detection and screening of specific primers and probes for highly virulent Klebsiella pneumoniae and Streptococcus pneumoniae

[0054] A pure culture of highly virulent Klebsiella pneumoniae 13383 (American Type Culture Collection) was placed in a 2 mL centrifuge tube, and 200 μL of DNA rapid extraction reagent was added using a micropipette. The rapid extraction reagent consists of Chelex-100, lysozyme, and Tris-EDTA buffer (10 mM Tris, 1 mM EDTA, pH 8.0), and the mass volume ratio of Chelex-100, lysozyme, and Tris-EDTA buffer is 5 g: 200 mg: 100 mL. Vortex for 30 seconds and fully resuspend. Then, the centrifuge tube was placed in a 37°C and 95°C water bath for 10 minutes and 15 minutes, respectively. The centrifuge tube after the water bath was placed in a centrifuge at 10,000 rpm for 5 minutes, and the supernatant liquid in the centrifuge tube was extracted using a 1 mL syringe. After filtering through a 0.45 μm filter membrane, DNA without impurities was obtained.

[0055] 2 μL upstream and downstream primers (1 μL each of upstream and downstream primers) and 0.5 μL probe of different combinations were added to 29.5 μL reaction buffer. The reaction buffer was composed of 20 mM Tris-HCl buffer (pH 8.8), 15 mM (NH4) 2SO4, 50 mM KCl, 5 mM MgSO4, 0.1 wt.% Tween-20, 1.5 mM dNTPs, 0.6 mg / mL BSA, 4 mM ATP, 30 mM creatine phosphate, 5.5% (w / v) polyethylene glycol 20000 and 2 mM dithiothreitol (DTT). Subsequently, 2 μL of DNA template was added, wherein the primer and probe concentrations were 10 μM, and the specific sequences of the primers and probes were as shown in Table 1.

[0056] Add 50 mg of freeze-dried enzyme powder (consisting of 50 μg / mL Bst DNA polymerase, 10 μg / mL Nt.BstNBI nuclease, 0.5 mg / mL single-stranded DNA binding protein (SSB), 500 ng / μL UvsX recombinase and 200 ng / μL creatine kinase) to the prepared reaction system, dissolve and mix thoroughly, and place in a constant temperature amplification detection device at 39°C; collect fluorescence signals every 30 seconds (the selection of the signal collection channel is consistent with the design of the fluorescent probe); the reaction time is 20 minutes.

[0057] Different primer combinations were screened according to the above reaction system and reaction conditions. The results are as follows: Figure 1 As shown. The results showed that different primer sets could amplify effectively, but the amplification effects were different. Among them, the IucAF2 / IucAR2-proe2 (F2R2) primer set showed nonspecific amplification, and the fluorescence signal appeared and started too early; the fluorescence appeared and started too early or too late in the IucAF1 / IucAR1-proe1 (F1R1) and IucAF4 / IucAR4-proe4 (F4R4) primer sets; only the IucAF3 / IucAR3-proe3 (F3R3) primer set showed a typical S-shaped amplification curve, and the amplification time and signal intensity were all in the ideal range. Finally, the lucAF3 / IucAR3 proe3 (F3R3) primer set with the best amplification efficiency was selected as the primer-probe combination for subsequent experiments.

[0058] Example 2: Real-time MIRA reaction specificity and sensitivity test for detecting highly virulent Klebsiella pneumoniae

[0059] 1. Reaction Specificity Detection

[0060] The DNA of the strains in Table 2 was rapidly extracted using the DNA extraction method according to the method of Example 1, and then the DNA templates of different strains were subjected to real-time fluorescent MIRA detection.

[0061] Table 2 List of positive control strains (highly virulent Klebsiella pneumoniae 13383) and negative control strains (other strains)

[0062]

[0063] A specificity experiment was conducted using a highly virulent Klebsiella pneumoniae genomic DNA and genomic DNA of five other bacteria (Escherichia coli, Salmonella, Pseudomonas aeruginosa, Staphylococcus aureus, Clostridium perfringens) as templates. The reaction system was the same as in Example 1, and the primer probe set used was lucAF3 / lucAR3 proe3 (F3R3). The results are as follows: Figure 2As shown. The results show that the primer-probe combination (lucAF3 / IucAR3 proe3) provided by the present invention is positive for both strains of highly virulent Klebsiella pneumoniae, and no detection is made for the other five bacteria. This result shows that the fluorescent MIRA method established based on the primer-probe set provided by the present invention has good specificity for highly virulent Klebsiella pneumoniae.

[0064] 2. Reaction sensitivity test

[0065] The DNA of the highly virulent Klebsiella pneumoniae 13383 strain was extracted according to the steps of Example 1, and the DNA template concentration was diluted 10 times to 1.68×10 0 pg / mL 1.68×10 5 pg / mL, respectively, and Real time MIRA amplification was performed, while ddH2O was used as NA, and finally the fluorescence PCR instrument was used to observe the test results. The test results are as follows Figure 3 The results showed that the detection limit of Real-time MIRA for the highly virulent Klebsiella pneumoniae 13383 strain was 1.68×10 0 pg / mL, with the increase of DNA concentration, the fluorescence signal of the amplification curve gradually increased, showing a good linear relationship and repeatability, which further verified the high sensitivity of the detection method provided by the present invention.

[0066] Example 3 Application of the detection method of Example 2 in actual samples (milk)

[0067] 1. Accuracy test

[0068] In order to verify the practicality of the method provided in Example 2 of the present invention in detecting highly virulent Klebsiella pneumoniae in actual samples, 50 portions of milk from cows with mastitis were taken. 1 mL of milk was centrifuged at 15000 rpm for 10 min, and the supernatant was discarded. The precipitated DNA was extracted using the DNA extraction method in Example 1. For the DNA template, conventional PCR detection and Real-time MIRA detection (Real-time MIRA detection method in Example 2) were performed respectively. The results showed that the detection results of Real-time MIRA were consistent with the results of ordinary PCR detection, and 21 of the 50 samples contained the iucA gene, indicating that 42% of the milk was contaminated with highly virulent Klebsiella pneumoniae.

[0069] In addition, equal amounts of highly virulent Klebsiella pneumoniae 13383, Escherichia coli ATCC 25922, and Staphylococcus aureus ATCC 25923 were added to healthy milk, and the method of Example 2 was used for detection. At the same time, healthy milk without any strains was used as the NTC blank. The detection results are as follows: Figure 4The results show that when the primer-probe combination designed by the present invention is used to detect milk contaminated by different strains, only the milk sample with highly virulent Klebsiella pneumoniae has a higher fluorescence signal, while the other treatment groups and the blank control group have no obvious fluorescence signals, indicating that the primer-probe combination designed by the present invention also has accurate detection capabilities for actual samples.

[0070] 2. Sensitivity testing of actual samples

[0071] Take 10 mL of healthy milk sample and add 1×10 8 CFU of highly virulent Klebsiella pneumoniae 13383. Then, 1 mL of milk contaminated with highly virulent Klebsiella pneumoniae was diluted 10-fold with healthy milk, and 100 μL of the diluted contaminated milk was spread on a plate and the number of colonies was counted. The effective viable counts of highly virulent Klebsiella pneumoniae 13383 in samples with different dilutions were 23×10 5 CFU / mL, 16×10 4 CFU / mL, 18×10 3 CFU / mL, 11×10 2 CFU / mL, 9×10 1 CFU / mL and 8×10 0 CFU / mL. The diluted milk samples were subjected to DNA extraction and Real-time MIRA detection according to the method in Example 1 (Real-time MIRA detection method in Example 2), and healthy milk without the addition of highly virulent Klebsiella pneumoniae 13383 was used as NA. The results are shown in Figure 5 It shows that when the colony count in milk is 8×10 0 CFU / mL, the starting time of the amplification curve was greater than 30 cycles, indicating that the minimum detection limit of this method for highly virulent Klebsiella pneumoniae in actual samples was 8×10 0 CFU / mL.

[0072] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A primer-probe combination for real-time fluorescent MIRA detection of highly virulent Klebsiella pneumoniae, characterized in that: The primer-probe combination comprises an upstream primer whose nucleotide sequence is as described in SEQ ID No.7, a downstream primer whose nucleotide sequence is as described in SEQ ID No.8, and a probe whose nucleotide sequence is as described in SEQ ID No.

9.

2. Use of the primer-probe combination according to claim 1 in the preparation of a product for detecting and / or assisting in the detection of highly virulent Klebsiella pneumoniae.

3. The use according to claim 2, characterized in that The products include reagents and detection kits.

4. A product for detecting and / or assisting in the detection of highly virulent Klebsiella pneumoniae, characterized in that: The product comprises the primer-probe combination according to claim 1.

5. The product according to claim 4, characterized in that The product also includes a DNA rapid extraction reagent, a reaction buffer and a freeze-dried enzyme powder.

6. The product according to claim 5, characterized in that The rapid DNA extraction reagent comprises Chelex-100, lysozyme and Tris-EDTA buffer; the mass volume ratio of the Chelex-100, the lysozyme and the Tris-EDTA buffer is 5 g: 200 mg: 100 mL; The reaction buffer contains 20 mM Tris-HCl, 15 mM (NH4)2SO4, 50 mM KCl, 5 mM MgSO4, 0.1 wt.% Tween-20, 1.5 mM dNTPs, 0.6 mg / mL BSA, 4 mM ATP, 30 mM creatine phosphate, 5.5% (w / v) polyethylene glycol 20000 and 2 mM dithiothreitol; The freeze-dried enzyme powder includes 50 μg / mL Bst DNA polymerase, 10 μg / mL Nt.BstNBI endonuclease, 0.5 mg / mL single-stranded DNA binding protein, 500 ng / mL UvsX recombinase and 200 ng / μL creatine kinase.

7. The product according to claim 4, characterized in that The products include reagents and detection kits.

8. Use of the primer-probe combination of claim 1 or the product of any one of claims 4 to 7 in detecting highly virulent Klebsiella pneumoniae for non-diagnostic and therapeutic purposes.

9. A method for detecting highly virulent Klebsiella pneumoniae for non-diagnostic and non-therapeutic purposes, characterized in that: The following steps are involved: The DNA genome of the sample to be tested is used as the template DNA, the template DNA and the primer-probe combination of claim 1 are mixed and then subjected to a constant temperature reaction, and real-time fluorescence MIRA detection is performed; if fluorescence appears, it is determined that the sample to be tested contains highly virulent Klebsiella pneumoniae.

10. The method according to claim 9, characterized in that The temperature of the isothermal reaction is 37-42° C. and the time is 15-20 min.

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