Nucleic acid composition, detection system and detection device for rapidly detecting klebsiella pneumoniae and carbapenem drug resistance genes

Abstract

The invention relates to the technical field of biological detection, in particular to a nucleic acid composition, a detection system and a detection device for rapidly detecting klebsiella pneumoniae and carbapenem drug resistance genes. The invention relates to a nucleic acid composition for rapidly detecting klebsiella pneumoniae and carbapenem drug resistance genes. The nucleic acid composition comprises a one-step detection primer group combining isothermal amplification and CRISPR (clustered regularly interspaced short palindromic repeats), the invention discloses a detection system for rapidly detecting klebsiella pneumoniae and carbapenem drug resistance genes. The detection system comprises a nucleic acid composition, a loop-mediated isothermal amplification reaction reagent, a CRISP reaction reagent and an indicator, a detection device for rapidly detecting klebsiella pneumoniae and carbapenem drug resistance genes is characterized by comprising a mixing plate and a reaction plate, reaction is performed under the constant temperature condition, a reaction carrier is easy to obtain, a temperature cycle instrument which is high in value and tedious in step is not needed, and the detection cost is reduced. The reaction time is greatly shortened, and the detection efficiency is improved.

Description

Technical Field

[0001] The present application relates to the field of biological detection technology, and more specifically, to a nucleic acid composition, a detection system and a detection device for rapid detection of Klebsiella pneumoniae and carbapenem resistance genes. Background Art

[0002] Klebsiella pneumoniae is a serious nosocomial microorganism and a conditional pathogen with a high infection rate. Its persistent infection can cause severe pneumonia and irreversible damage to the human body. Carbapenems are currently the main drugs used to treat Klebsiella pneumoniae. However, in clinical practice, due to the extensive use of antibiotics, drug resistance continues to increase, and the sensitivity of β-lactamase drugs such as imipenem and meropenem has gradually decreased, resulting in a large increase in carbapenem-resistant Klebsiella pneumoniae in clinical practice. The detection of carbapenem-resistant Klebsiella pneumoniae is currently mainly based on culture methods. Because it takes a long time to culture and cannot be reported in a timely manner, a rapid and accurate detection method is urgently needed. Summary of the invention

[0003] In order to address the technical problem of slow detection of Klebsiella pneumoniae and carbapenem resistance genes in the prior art, the present application provides a nucleic acid composition, a detection system and a detection device for rapid detection of Klebsiella pneumoniae and carbapenem resistance genes.

[0004] In the first aspect, the present application provides a nucleic acid composition for rapid detection of Klebsiella pneumoniae and carbapenem resistance genes, using the following technical solution:

[0005] A nucleic acid composition for rapid detection of Klebsiella pneumoniae and carbapenem resistance genes, comprising an isothermal amplification combined with a CRISPR one-step detection primer set.

[0006] Loop-mediated isothermal amplification is a technology invented by Japanese scientists around 2000. It is achieved by identifying the 4-6 segments in the sequence of about 200bp of the template sequence. The strand displacement enzyme plays a strand displacement role to form the core element of loop-mediated isothermal amplification and form exponential amplification. The loop-mediated isothermal amplification technology has simple primer design, convenient operation, low machine requirements, and can even react in a water bath, which is very suitable for real-time clinical detection. Real-time fluorescence detection can be performed by nucleic acid dyes, and the reaction progress can be judged from the Ct value of the fluorescence curve. Other visual dyes can also be used for detection, and color changes in visible light will occur. The technology has a lot of selectivity and is very suitable for the requirements of clinical real-time detection. The diagnosis report can be directly issued within half an hour, greatly compressing the time consumed by diagnosis. The full name of CRISPR is regularly interspaced short palindromic repeats. This type of technology uses different types of Cas proteins and their corresponding guide RNAs to combine and cut to mediate the detection of nucleic acids. The detection technology of loop-mediated isothermal amplification combined with CRISPR can improve the stability and sensitivity of the detection, and at the same time achieve the purpose of multiple detection of pathogens.

[0007] Preferably, the isothermal amplification combined with CRISPR one-step detection primer set includes a Klebsiella pneumoniae phoE gene primer set and a carbapenem-resistant KPC-2 gene primer set;

[0008] The Klebsiella pneumoniae phoE gene primer set includes:

[0009] The upstream external primer F3 is shown in SEQ ID No. 1;

[0010] Downstream external primer B3 is shown as SEQ ID No.2;

[0011] The upstream internal primer FIP is shown as SEQ ID No. 3;

[0012] The downstream internal primer BIP is shown as SEQ ID No. 4;

[0013] The upstream circularization primer LF is shown in SEQ ID No.5;

[0014] The upstream cyclization primer LB is shown in SEQ ID No.6;

[0015] The carbapenem-resistant KPC-2 gene primer set includes:

[0016] The upstream external primer F3 is shown in SEQ ID No.7;

[0017] Downstream external primer B3 is shown as SEQ ID No.8;

[0018] The upstream internal primer FIP is shown as SEQ ID No.9;

[0019] The downstream internal primer BIP is shown as SEQ ID No. 10;

[0020] The upstream circularization primer LF is shown in SEQ ID No.11;

[0021] The upstream circularization primer LB is shown as SEQ ID No.12.

[0022] In a second aspect, the present application provides a detection system for rapid detection of Klebsiella pneumoniae and carbapenem resistance genes.

[0023] A detection system for rapid detection of Klebsiella pneumoniae and carbapenem resistance genes, comprising the above-mentioned nucleic acid composition, a loop-mediated isothermal amplification reaction reagent, a CRISPR reaction reagent and an indicator.

[0024] Preferably, the loop-mediated isothermal amplification reaction reagents include Bst DNA polymerase, dNTP mixture, Tris-HCl, 10mM (NH4)2SO4, KCl, Tween20 and MgSO4.

[0025] Preferably, the CRISPR reaction reagent includes Cas protein and guide RNA.

[0026] Preferably, the guide RNA includes the Klebsiella pneumoniae phoE gene guide RNA as shown in SEQ ID No.20 and the carbapenem-resistant KPC-2 gene guide RNA as shown in SEQ ID No.21.

[0027] Preferably, an internal reference quality control nucleic acid composition is also included:

[0028] The upstream external primer F3 is shown as SEQ ID No. 13;

[0029] Downstream external primer B3 is shown in SEQ ID No.14;

[0030] The upstream internal primer FIP is shown as SEQ ID No. 15;

[0031] The downstream internal primer BIP is shown as SEQ ID No. 16;

[0032] The upstream circularization primer LF is shown in SEQ ID No.17;

[0033] The upstream circularization primer LB is shown as SEQ ID No.18.

[0034] Preferably, the indicator comprises a fluorescent probe as shown in SEQ ID No.19.

[0035] In a third aspect, the present application provides a detection device for rapid detection of Klebsiella pneumoniae and carbapenem resistance genes.

[0036] A detection device for rapid detection of Klebsiella pneumoniae and carbapenem resistance genes, comprising a mixing plate and a reaction plate, wherein the mixing plate is provided with a sample mixing part and a reaction part, and the reaction plate is provided with a connecting hole, wherein one end of the connecting hole corresponds to the sample mixing part, and the other end of the connecting hole corresponds to the reaction part.

[0037] Preferably, it also includes a valve for connecting or closing the reaction part.

[0038] In summary, this application has the following beneficial effects:

[0039] 1. Six primers and six primers were designed for the phoE gene and carbapenem-resistant KPC-2 gene of Klebsiella pneumoniae, respectively. The amplified products are highly specific. At the same time, corresponding guide RNAs are designed for the corresponding primers, which can specifically identify the sequence of the amplified products and improve the specificity of the reaction. At the same time, internal reference genes are designed in each reaction to control the quality of the samples. The double amplification detection design can accurately control the reaction.

[0040] 2. The detection of this application is carried out under constant temperature conditions, and the reaction carrier is easily available, and no expensive and complicated temperature cycle instrument is required, which reduces the detection cost. The reaction time is greatly shortened, and the detection efficiency is improved.

[0041] 3. The acrylic material detection device of the present application has the advantage of real-time detection, and the detection of pathogens is convenient, fast, and low-cost, and is suitable for rapid screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is an overall schematic diagram of the detection device of this application;

[0043] Figure 2 This is a schematic diagram of the explosion of the detection device of this application;

[0044] Figure 3 A schematic diagram of the valve of the detection device of this application;

[0045] Figure 4 A top view of the control panel of the detection device of the present application;

[0046] Figure 5 A top view of a sample adding plate of the detection device of the present application;

[0047] Figure 6 A top view of a mixing plate of the detection device of the present application;

[0048] Figure 7 This is a schematic diagram of the valve opening state of the detection device of this application;

[0049] Figure 8 This is a schematic diagram of the valve closing state of the detection device of this application.

[0050] Fig. 9 A top view of a reaction plate of the detection device of the present application;

[0051] Figure numerals: 1. control plate; 2. sample adding plate; 3. mixing plate; 4. reaction plate; 5. valve; 6. valve head; 7. valve core; 8. control hole; 9. sample adding hole; 10. rotating hole; 11. sample mixing part; 12. reaction part; 13. sample retention hole; 14. mixing hole; 15. sample mixing hole; 16. C-shaped connecting hole; 17. reaction hole; 18. connecting hole. DETAILED DESCRIPTION

[0052] A nucleic acid composition for rapid detection of Klebsiella pneumoniae and carbapenem resistance genes, comprising an isothermal amplification combined with a CRISPR one-step detection primer set.

[0053] Specifically, primer sets were designed based on the Klebsiella pneumoniae phoE gene and carbapenem-resistant KPC-2 gene sequences in NCBI (NC_016845.1 and NG_049253.1), and then the corresponding guide RNAs were designed.

[0054] Among them, the Tm value of the external primer set should be 55-63℃, and the base length is 15-25. The Tm value of the 20 bases at the 5' end of the internal primer set should be higher than the Tm value of other primer sets, and is controlled at 60-68℃. The Tm value of the 20 bases at the 3' end of the internal primer set is 55-63℃. The GC content of all primers is controlled at 30%-65%. The stability of the 5' and 3' ends of the primers needs to be ensured, and the formation of dimers between the primers themselves and between primers needs to be avoided. There are strict requirements for the position of the primer anchor on the target sequence. The base length between the external primer sets is controlled at 160-220, and the corresponding bases between the internal primers and the external primers are controlled within 20, and the base length between the internal primer sets is controlled at 120-180. These 4-6 primers can be anchored at 6-8 positions of the nucleic acid, relying on the strand displacement effect of the external primer group and the complementary sequence characteristics of the 5' end of the internal primer group and the template to form an amplification element that can amplify the signal, forming a series of amplification products of different lengths.

[0055] The guide RNA sequence is designed for the amplified fragment of the loop-mediated primer. The spacer sequence of the guide RNA needs to be paired with the F2-B2 region of the loop-mediated amplification, and there needs to be a TTN base recognition site before the pairing site. The length of the spacer sequence is 16-24 bases, with 20 bases being the best. It is necessary to ensure that the spatial structure of the entire guide RNA is correct, and the guide RNA formed by the spacer sequence and the fixed sequence can form three stem loops. At the same time, it is necessary to ensure that there is no interference between the guide RNA sequence and the loop-mediated amplification primer, and no polymers are formed. The 5' end of the guide RNA is a species-specific sequence, which is an RNA of about 85 bases in length, forming a three-segment stem-loop structure; the 3' end sequence is a template recognition sequence, which is an RNA of 18-24 bases in length, and the site for the guide RNA to recognize the template is NTTN.

[0056] The present invention designs 6 specific primers for the phoE gene of Klebsiella pneumoniae and the carbapenem-resistant KPC-2 gene fragment respectively.

[0057] The Klebsiella pneumoniae phoE gene primer set includes:

[0058] The upstream external primer F3 is shown in SEQ ID No. 1;

[0059] Downstream external primer B3 is shown as SEQ ID No.2;

[0060] The upstream internal primer FIP is shown as SEQ ID No. 3;

[0061] The downstream internal primer BIP is shown as SEQ ID No. 4;

[0062] The upstream circularization primer LF is shown in SEQ ID No.5;

[0063] The upstream cyclization primer LB is shown as SEQ ID No.6;

[0064] The primer set for carbapenem-resistant KPC-2 gene includes:

[0065] The upstream external primer F3 is shown in SEQ ID No.7;

[0066] Downstream external primer B3 is shown as SEQ ID No.8;

[0067] The upstream internal primer FIP is shown as SEQ ID No.9;

[0068] The downstream internal primer BIP is shown as SEQ ID No. 10;

[0069] The upstream circularization primer LF is shown in SEQ ID No.11;

[0070] The upstream circularization primer LB is shown in SEQ ID No.12;

[0071] The guide RNA of the Klebsiella pneumoniae phoE gene is shown in SEQ ID No.20, and the guide RNA of the carbapenem-resistant KPC-2 gene is shown in SEQ ID No.21.

[0072] A detection system for rapid detection of Klebsiella pneumoniae and carbapenem resistance genes, comprising the above-mentioned nucleic acid composition, a loop-mediated isothermal amplification reaction reagent, a CRISP reaction reagent, an internal reference quality control nucleic acid composition and an indicator.

[0073] Among them, the concentration of the above-mentioned outer primer F3 is 0.2μM, the concentration of the outer primer B3 is 0.2μM, the concentration of the inner primer FIP is 1.6μM, the concentration of the inner primer BIP is 1.6μM, the concentration of the circularization primer LF is 0.8μM, and the concentration of the circularization primer LB is 0.8μM; the concentration of the guide RNA corresponding to the primer set is 30nM.

[0074] The loop-mediated isothermal amplification reaction reagents include Bst DNA polymerase, dNTP mixture, ris-HCl, 10mM (NH4)2SO4, KCl, Tween20 and MgSO4. Specifically, Bst DNA polymerase 8U, 30nM Cas12b, 1mM dNTP, 20mM Tris-HCl, 10mM (NH4)2SO4, 50mM KCl, 0.1% Tween20 and 2mM MgSO4.

[0075] The CRISPR reaction reagent includes a Cas protein and the above-mentioned guide RNA. The Cas protein is Cas12b or a high-temperature resistant Cas protein having a cis-cutting single-stranded mediated trans-cutting activity similar to that of Cas12b, which can be derived from FnCas12a, AsCas12a, LbCas12a, AacCas12b, Cas14 or AapCas12b; AapCas12b is preferred in this method.

[0076] The concentration of Cas12b is 50-250nM, the concentration of guide RNA is 50-250nM, and the ratio between Cas12b and guide RNA is between 1:1-1:1.5.

[0077] The internal reference quality control nucleic acid composition was designed based on the human gene ACTB gene sequence (NG_007992.1) in NCBI, and 6 specific primers were specifically included:

[0078] The upstream external primer F3 is shown as SEQ ID No. 13;

[0079] Downstream external primer B3 is shown in SEQ ID No.14;

[0080] The upstream internal primer FIP is shown as SEQ ID No. 15;

[0081] The downstream internal primer BIP is shown as SEQ ID No. 16;

[0082] The upstream circularization primer LF is shown in SEQ ID No.17;

[0083] The upstream circularization primer LB is shown as SEQ ID No.18.

[0084] The indicator includes a target gene indicator and an internal reference quality control indicator, wherein the target gene primer indicator is a fluorescent probe including a single-stranded DNA sequence with a sequence length of 6-20, a 5' end fluorescent group can be FAM, VIC or ROX, and a 3' end quenching group can be DAB or BHQ. The present application prefers a probe with a length of 6 bases, a 5' end labeled with ROX, a 3' end labeled with BHQ, and a concentration of 1 μM, as shown in SEQ ID No. 19. The concentration of the fluorescent probe is 500nM-1μM.

[0085] The internal reference quality control indicator is a fluorescent dye, which can be SYTO 9, SYTO16, SYBR Green or Eva Green. SYBR Green is preferred in this application, and the concentration is 10×.

[0086] A lyophilized protective agent may also be added to the detection system to form a temperature amplification combined with CRISPR one-step detection of all components of the lyophilized ball. The lyophilized protective agent may be trehalose, sucrose or mannitol, such as trehalose concentration of 10%-20%, sucrose concentration of 20mM-80mM, and mannitol concentration of 100mM-160mM. Specifically, the lyophilized protective agent is trehalose.

[0087] In addition, the internal reference quality control nucleic acid composition can also be formed into a freeze-dried ball, which can be a single freeze-dried ball or freeze-dried in combination with the target gene primers. It should be noted that the target gene in the present application is the Klebsiella pneumoniae phoE gene primer set and the carbapenem-resistant KPC-2 gene primer set.

[0088] Reference Figure 1 and Figure 2 A detection device for rapid detection of Klebsiella pneumoniae and carbapenem resistance genes, which includes a control plate 1, a sample plate 2, a mixing plate 3, and a reaction plate 4 from top to bottom. The control plate 1, the sample plate 2, the mixing plate 3 and the reaction plate 4 are fixed with double-sided tape, and the bottom of the reaction plate 4 is sealed with a sealing strip. A valve 5 is also included.

[0089] Reference Figure 3-Figure 5 , the valve 5 includes a valve head 6 and a valve core 7. The control board 1 is provided with a control hole 8, and the valve head 6 passes through the control hole 8. A sample adding hole 9 is provided on one side of the sample adding plate 2 extending out of the control board 1. In the present application, two sample adding holes 9 are provided, one of which is used to add the sample to be tested, and the other is used to add the sample releasing agent. The sample adding plate 2 is provided with a rotating hole 10 corresponding to the control hole 8, and the valve core 7 is passed through the rotating hole 10. In addition, the circumference of the valve core 7 is larger than the circumference of the control hole 8, so the valve core 7 is positioned between the control board 1 and the sample adding plate 2. In addition, in order to increase the connection strength of the valve core 7, it can be fixed by means of screws or the like, that is, when the screws are tightened, the valve core 7 cannot rotate freely, and when the screws are loosened, the valve core 7 can rotate freely.

[0090] Reference Figure 6 The mixing plate 3 is provided with a sample mixing part 11 and a reaction part 12. The sample mixing part 11 corresponds to the sample adding hole 9, and the reaction part 12 corresponds to the rotating hole 10. Specifically, the sample mixing part 11 is provided with a sample retention hole 13 and a mixing hole 14, wherein two sample retention holes 13 are provided, and the sample retention holes 13 correspond to the sample adding hole 9, and one mixing hole 14 is provided, and the mixing hole 14 is connected with the sample retention hole 13 in a Y shape. The reaction part 12 is provided with two sample mixing holes 15 and a C-shaped connecting hole 16, and the two sample mixing holes 15 are connected through the C-shaped connecting hole 16, and the C-shaped connecting hole 16 corresponds to the rotating hole 10.

[0091] Reference Figure 7 and Figure 8 , the valve core 7 is placed in the C-shaped communication hole 16, and the shape of the valve core 7 is adapted to the C-shaped communication hole 16 and can rotate in the C-shaped communication hole 16. When the valve core 7 is closed, the valve core 7 is placed at the connection between the C-shaped communication hole 16 and the mixing hole 15. That is, the liquid cannot enter the mixing hole 15 through the C-shaped communication hole 16; when the valve core 7 is opened, the valve core 7 is placed at one end of the C-shaped communication hole 16, and at this time, the C-shaped communication hole 16 is connected with both mixing holes 15.

[0092] Reference Figure 8 and Fig. 9 , the reaction plate 4 is also provided with two sample retention holes 13 and two reaction holes 17. The number of reaction holes 17 is provided according to the number of test targets. It is set according to the reaction requirements and is used to store isothermal reaction freeze-dried balls. In the present application, two reaction holes 17 are provided, each of which contains a freeze-dried ball. One of the freeze-dried balls contains a Klebsiella pneumoniae detection target system and an internal standard system, and the other freeze-dried ball contains a carbapenem-resistant detection target system and an internal standard system. The two reaction holes 17 correspond one-to-one to the two mixed sample holes 15; the reaction plate 4 is also provided with a connecting hole 18, one end of the connecting hole 18 corresponds to the mixing hole 14, and the other end of the connecting hole 18 corresponds to and is connected to the C-shaped connecting hole 16.

[0093] Specifically, add the sample to be tested to the sample well 9, add the sample release agent to another sample well 9, and seal the sample well 9 with a transparent tape. At this time, the sample to be tested and the sample release agent enter the sample retention well 13 respectively, and enter the mixing well 14 through the Y-shaped channel for mixing. Turn the valve head 6 to open the valve core 7, and swing the detection device. During the continuous swinging process, the sample enters the other end of the connecting hole 18 from one end of the connecting hole 18, and enters the C-shaped connecting hole 16, enters the mixing well 15 from the C-shaped connecting hole 16, and finally enters the reaction well 17 to react with the preset isothermal reaction freeze-dried ball. Then turn the valve head 6 again to close the valve core 7.

[0094] Detection test

[0095] 1. Use sputum samples to detect the phoE gene and carbapenem-resistant KPC-2 gene of Klebsiella pneumoniae.

[0096] 1. Extraction of nucleic acid

[0097] The sputum sample was pretreated with 4% NaOH, diluted and added to the sample well 9, a sample release agent was added to another sample well 9, the sample well 9 was sealed with a transparent tape, the valve head 6 was turned to close the valve core 7, the detection device was manually shaken to mix the sample release agent and the pretreated sample, and incubated for 15 minutes;

[0098] 2. Isothermal testing

[0099] Turn the valve head 6 to open the valve core 7, shake the chip manually to allow the mixed sample to re-dissolve the freeze-dried balls in the two reaction wells 17, turn the valve head 6 to close the valve core 7, react at 63°C for 30-45 minutes, and record the fluorescence signal in real time.

[0100] 3. Result determination

[0101] Whether the reaction occurs is determined based on the fluorescence signal. The specific judgment criteria are shown in Table 1.

[0102] Table 1 Reaction judgment criteria

[0103]

[0104] 2. The sensitivity and specificity of loop-mediated isothermal amplification combined with CRISPR in detecting the phoE gene and carbapenem-resistant KPC-2 gene of Klebsiella pneumoniae.

[0105] The extracted carbapenem-resistant Klebsiella pneumoniae (confirmed to contain KPC-2 gene, clinically more than 90% contain) nucleic acid was quantified to 105 copies / μL, then diluted to 104 copies / μL, 103 copies / μL, 102 copies / μL, 101 copies / μL and 100 copies / μL in sequence, added to the reaction chip, and its sensitivity was detected (see Table 2 for the judgment criteria), and compared with the fluorescent PCR probe method. The results are shown in Table 2. Table 2 Sensitivity of loop-mediated isothermal amplification combined with CRISPR to detect the phoE gene and carbapenem-resistant KPC-2 gene of Klebsiella pneumoniae.

[0106]

[0107] The extracted nucleic acids of Staphylococcus aureus, Haemophilus influenzae, Pseudomonas aeruginosa, Streptococcus pneumoniae, Group A Streptococcus, Catmorella, Acinetobacter baumannii, Legionella pneumophila and Mycobacterium tuberculosis were quantified to 105 copies / μL and added to the reaction chips respectively to detect their specificity. The results are shown in Table III.

[0108] Table III Specificity of loop-mediated isothermal amplification combined with CRISPR for detecting the phoE gene and carbapenem-resistant KPC-2 gene of Klebsiella pneumoniae.

[0109]

[0110]

[0111] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed, but such modifications are protected by patent law as long as they are within the scope of the claims of the present application.

Claims

1. A nucleic acid composition for rapid detection of Klebsiella pneumoniae and carbapenem resistance genes, characterized in that: Includes isothermal amplification combined with a one-step CRISPR detection primer set.

2. The nucleic acid composition for rapid detection of Klebsiella pneumoniae and carbapenem resistance genes according to claim 1, characterized in that: The isothermal amplification combined with CRISPR one-step detection primer set includes a Klebsiella pneumoniae phoE gene primer set and a carbapenem-resistant KPC-2 gene primer set; The Klebsiella pneumoniae phoE gene primer set includes: The upstream external primer F3 is shown in SEQ ID No. 1; Downstream external primer B3 is shown as SEQ ID No.2; The upstream internal primer FIP is shown as SEQ ID No. 3; The downstream internal primer BIP is shown as SEQ ID No. 4; The upstream circularization primer LF is shown in SEQ ID No.5; The upstream cyclization primer LB is shown as SEQ ID No.6; The carbapenem-resistant KPC-2 gene primer set includes: The upstream external primer F3 is shown in SEQ ID No.7; Downstream external primer B3 is shown as SEQ ID No.8; The upstream internal primer FIP is shown as SEQ ID No.9; The downstream internal primer BIP is shown as SEQ ID No. 10; The upstream circularization primer LF is shown in SEQ ID No.11; The upstream circularization primer LB is shown as SEQ ID No.

12.

3. A detection system for rapid detection of Klebsiella pneumoniae and carbapenem resistance genes, characterized in that: It comprises the nucleic acid composition according to claim 1, a loop-mediated isothermal amplification reaction reagent, a CRISPR reaction reagent and an indicator.

4. The detection system for rapid detection of Klebsiella pneumoniae and carbapenem resistance genes according to claim 3, characterized in that: The loop-mediated isothermal amplification reaction reagents include Bst DNA polymerase, dNTP mixture, Tris-HCl, 10mM (NH4)2SO4, KCl, Tween20 and MgSO4.

5. The detection system for rapid detection of Klebsiella pneumoniae and carbapenem resistance genes according to claim 3, characterized in that: The CRISPR reaction reagent includes Cas protein and guide RNA.

6. The detection system for rapid detection of Klebsiella pneumoniae and carbapenem resistance genes according to claim 5, characterized in that: The guide RNA includes the Klebsiella pneumoniae phoE gene guide RNA as shown in SEQ ID No.20 and the carbapenem-resistant KPC-2 gene guide RNA as shown in SEQ ID No.

21.

7. The detection system for rapid detection of Klebsiella pneumoniae and carbapenem resistance genes according to claim 3, characterized in that: Also included is an internal reference quality control nucleic acid composition: The upstream external primer F3 is shown as SEQ ID No. 13; Downstream external primer B3 is shown in SEQ ID No.14; The upstream internal primer FIP is shown as SEQ ID No. 15; The downstream internal primer BIP is shown as SEQ ID No. 16; The upstream circularization primer LF is shown in SEQ ID No.17; The upstream circularization primer LB is shown as SEQ ID No.

18.

8. The detection system for rapid detection of Klebsiella pneumoniae and carbapenem resistance genes according to claim 3, characterized in that: The indicator includes a fluorescent probe as shown in SEQ ID No.

19.

9. A detection device for rapid detection of Klebsiella pneumoniae and carbapenem resistance genes, characterized in that: The invention comprises a mixing plate (3) and a reaction plate (4), wherein the mixing plate (3) is provided with a sample mixing part (11) and a reaction part (12), and the reaction plate (4) is provided with a connecting hole (18), one end of the connecting hole (18) corresponds to the sample mixing part (11), and the other end of the connecting hole (18) corresponds to the reaction part (12).

10. The detection device for rapid detection of Klebsiella pneumoniae and carbapenem resistance genes according to claim 9, characterized in that: It also includes a valve (5) for connecting or closing the reaction part (12).

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