Primer group, composition, kit and device for detecting klebsiella pneumoniae

Abstract

The invention relates to a primer group, a composition, a kit and a device for detecting klebsiella pneumoniae. The composition comprises an upstream primer and a downstream primer of target double-stranded DNA, a TaqMan fluorescent probe, recombinase, T7RNA polymerase, transcriptase, ATP energy regeneration enzyme, melting enzyme, single-stranded binding protein and cutting enzyme. According to the invention, nucleic acid amplification enzyme and signal amplification enzyme are integrated together, the purpose of amplifying target double-stranded DNA to 109 times within 10-30 minutes under the condition of constant temperature of 25 DEG C is realized, and each nucleic acid amplification product can generate more than 10,000 fluorescence signals under the condition of constant temperature of 30 DEG C, so that efficient and rapid detection of samples is realized; according to the method, the omission ratio caused by false negative is extremely low, the operation is convenient, and the steps are simple.

Description

Technical Field

[0001] The present invention relates to the technical field of bacterial detection, and in particular to a primer set, a composition, a kit and a device for detecting Klebsiella pneumoniae. Background Art

[0002] Klebsiella pneumoniae (K. Pneumoniae) is a conditional pathogen. In rodents (such as mice and rats), infection with K. pneumoniae can cause pneumonia. Sick animals will experience symptoms such as lethargy, decreased activity, and shortness of breath. The lungs will become inflamed, and the alveolar structure will be destroyed, resulting in impaired gas exchange function. The main hazard to experimental animals is that when the infected animals are subjected to radiation, experimental burns, immunosuppression, etc., sepsis may be induced, leading to the death of experimental animals and affecting the normal progress of the experiment. In addition, this infection may cause changes in the physiological indicators of experimental animals, seriously affect the quality of experimental animals, and interfere with experimental results, such as causing changes in blood composition and abnormal immune responses.

[0003] According to the national standard for experimental animals in my country (GB 14922-2022 Laboratory Animal Microorganisms, Parasites Grades and Monitoring), specific pathogen-free (SPF) experimental rats and mice must exclude Klebsiella pneumoniae. The results of laboratory animal quality monitoring at home and abroad in recent years have also shown that the positive rate of Klebsiella pneumoniae in experimental rats and mice is relatively high, which will affect the quality of experimental animals and interfere with experimental results. According to the current national standard (GB / T 14926.17-2001 Laboratory Animal Klebsiella pneumoniae Detection Method), the detection of Klebsiella pneumoniae requires the isolation, culture and biochemical identification of samples. This method takes at least 3 days, the detection cycle is long, the operation is cumbersome, time-consuming and laborious. If the DNA amplification method is used, although Klebsiella pneumoniae can be detected in a relatively fast time, it still faces many problems.

[0004] At present, the methods of DNA amplification include conventional PCR and isothermal PCR. These technologies can quickly amplify the target DNA by 1 million times or even higher in a short time. Since its introduction in 1983, conventional PCR has relied on three-temperature (melting, annealing, extension) cycles, and has very high requirements for the stability of PCR instruments. Although two-temperature cycles can shorten the reaction time, the activity of DNA polymerase is not at the optimal temperature, so only shorter fragments can be amplified, and the time consumption has not been greatly reduced. Isothermal PCR relies on a stable temperature higher than room temperature (for example, 60°C), and still requires an instrument with precise temperature control. General room-temperature PCR relies on recombinase and polymerase with chain displacement to amplify shorter sequences, requiring an ATP regeneration system, which makes the reaction system more complicated and easily induces side reactions, interfering with the detection effect. Since the performance and operation mode of molecular diagnostic reagents are determined by their core enzyme components, new room-temperature nucleic acid amplification detection can be performed based on enzyme-mediated dual exponential amplification technology.

[0005] In the testing of experimental animals, the most common practice is to detect Klebsiella pneumoniae in experimental animals through isolation, culture and biochemical identification of bacteria. Although this method is the gold standard for pathogenic microorganism detection, its detection cycle is long and the operation is cumbersome. At the same time, the professional level of the tester has a great influence on the results of biochemical culture.

[0006] Compared with traditional biochemical testing, PCR testing by amplifying Klebsiella pneumoniae DNA is not only highly sensitive but can also help testing agencies determine whether Klebsiella pneumoniae is present in samples in a relatively short period of time. For experimental animal pathogens, the current common nucleic acid detection methods include ordinary PCR detection and constant temperature PCR detection. With the improvement of laboratory hardware conditions and the professional level of personnel, in addition to PCR detection methods, some group standards have begun to use constant temperature detection methods such as loop-mediated isothermal amplification (LAMP) and reverse transcription-loop-mediated isothermal amplification (RT-LAMP) to detect some experimental animal pathogens.

[0007] Although the common / constant temperature PCR detection methods have shortened the detection time of pathogenic microorganisms in experimental animals to a certain extent, they still face the problems of large investment in laboratory hardware and the need for professional personnel to operate. At the same time, there is still room for improvement in detection time. These all require experimental animal institutions to invest a large amount of costs, among which hardware conditions and environmental requirements directly affect the judgment of the results. Summary of the invention

[0008] In view of the problems in the prior art that the detection of Klebsiella pneumoniae takes a long time and requires professional equipment and professional personnel to operate, the present invention provides a primer set, a composition, a kit, and a device for detecting Klebsiella pneumoniae. The present invention can carry out detection at any location, has a simple operation process, is suitable for use by anyone, and can detect Klebsiella pneumoniae in a sample within 30 minutes at 25°C.

[0009] The technical solution provided by the present invention is specifically as follows:

[0010] In a first aspect, the present invention provides a primer set for RAA amplification of Klebsiella pneumoniae nucleic acid, comprising an upstream primer and a downstream primer of a target double-stranded DNA;

[0011] The nucleotide sequence of the upstream primer is shown in SEQ ID NO.: 5;

[0012] The nucleotide sequence of the downstream primer is shown in SEQ ID NO.:7.

[0013] In a second aspect, the present invention provides a Klebsiella pneumoniae detection composition, the Klebsiella pneumoniae detection composition comprising:

[0014] An upstream primer and a downstream primer of the target double-stranded DNA, wherein the upstream primer comprises a T7 promoter;

[0015] A recombinase that can catalyze the formation of a double-stranded DNA containing a T7 promoter by an upstream primer and a first single-stranded target double-stranded DNA;

[0016] T7 RNA polymerase, which is used to catalyze the transcription of double-stranded DNA containing a T7 promoter to generate single-stranded RNA;

[0017] Transcriptase, which catalyzes the reverse transcription of single-stranded RNA into double-stranded DNA / RNA;

[0018] ATP energy regenerating enzyme, which is used to provide ATP energy in DNA recombination and transcription processes;

[0019] Helicase, which is used to catalyze the hydrolysis of DNA / RNA double strands into RNA single strands and DNA single strands;

[0020] Single-strand binding protein, which is used to bind to single-stranded DNA and prevent the self-polymerization of single-stranded DNA;

[0021] TaqMan fluorescent probe, which includes an RNA chain that can bind to a single-stranded DNA, and the 5' end and 3' end of the RNA chain have a fluorescent group and a quenching group respectively;

[0022] A cleavage enzyme, which is used to cleave the RNA chain of the TaqMan fluorescent probe to release the fluorescent group when the TaqMan fluorescent probe is bound to the DNA single chain;

[0023] in:

[0024] The nucleotide sequence of the upstream primer is shown in SEQ ID NO.: 5;

[0025] The nucleotide sequence of the downstream primer is shown in SEQ ID NO.: 7;

[0026] The nucleotide sequence of the TaqMan fluorescent probe is shown in SEQ ID NO.:15.

[0027] In some embodiments, the quenching group of the TaqMan fluorescent probe is one of BHQ, BHQ1, BHQ2, and TAMRA, and the fluorescent group is one of FAM, FAM-X, CY3, CY5, HEX, and TET.

[0028] In some embodiments, the Klebsiella pneumoniae detection composition further comprises a plasmid standard of the target double-stranded DNA.

[0029] In some embodiments, the Klebsiella pneumoniae detection composition is used to amplify and detect target double-stranded DNA at a constant temperature of 25°C.

[0030] In some embodiments, the Klebsiella pneumoniae detection composition provided by the present invention further comprises a plasmid standard of the Klebsiella pneumoniae UreA gene fragment.

[0031] In a third aspect, the present invention provides a Klebsiella pneumoniae detection kit, which comprises the Klebsiella pneumoniae detection composition.

[0032] In some embodiments, the Klebsiella pneumoniae detection kit provided by the present invention further comprises a lysis solution, wherein the lysis solution comprises a broad-spectrum protease and a physiological buffer.

[0033] In some embodiments, the Klebsiella pneumoniae detection kit provided by the present invention comprises a lyophilization protectant, and each component in the Klebsiella pneumoniae detection composition is packaged in the form of lyophilized powder.

[0034] In a fourth aspect, the present invention provides a Klebsiella pneumoniae detection device, comprising:

[0035] A DNA extraction module, which is used to extract sample DNA;

[0036] An amplification module, which is used to amplify the sample DNA using the Klebsiella pneumoniae detection composition to obtain an amplification product;

[0037] The fluorescence detection module is used to detect the fluorescence signal of the amplification product.

[0038] The Klebsiella pneumoniae detection device also includes a judgment module:

[0039] The judgment module judges whether the sample contains Klebsiella pneumoniae according to the fluorescent signal; if there is no fluorescent signal, it is judged that the sample does not contain Klebsiella pneumoniae; if there is a fluorescent signal, it is judged that the sample contains Klebsiella pneumoniae.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] 1. The DNA extraction steps provided by the present invention are simplified, and the DNA extraction work can be completed by only adding a lysis solution and performing a 10-minute heat treatment.

[0042] 2. The enzyme-mediated room-temperature nucleic acid amplification technology for detecting Klebsiella pneumoniae of the present invention can achieve target nucleic acid amplification to 10 within 10-30 minutes at a constant temperature of 25°C by integrating nucleic acid amplification enzyme group amplification. 9 By integrating the signal amplification enzyme system, it can be achieved that one nucleic acid amplification product can generate more than 10,000 fluorescent signals under the constant temperature of 25°C, ultimately achieving efficient and rapid detection of samples.

[0043] 3. The present invention performs molecular detection of Klebsiella pneumoniae with high specificity and a sensitivity as low as 1 copy / μL, thereby enabling early detection of samples infected with Klebsiella pneumoniae.

[0044] 4. The present invention can be used in conjunction with a portable metal bath and a portable fluorescence detector, and can be carried to any location such as rural sites for detection, meeting application scenarios such as environmental detection within experimental barriers and detection next to animal cages.

[0045] 5. The present invention has a simple operation process, is suitable for use by anyone, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is a schematic diagram of the principle of detecting Klebsiella pneumoniae according to the present invention.

[0047] Figure 2-5 The TaqMan fluorescent probe screening results of Example 1 of the present invention;

[0048] Figure 6 The downstream primer screening result of Example 2 of the present invention is shown in FIG.

[0049] Figure 7 The upstream primer screening result of Example 3 of the present invention is shown in FIG.

[0050] Figure 8 The specific experimental results of Example 4 of the present invention;

[0051] Figure 9-10The sensitivity test results of Example 5 of the present invention are shown below:

[0052] Figure 11-12 The test results of the experimental example of the present invention are shown in FIG. DETAILED DESCRIPTION

[0053] The present invention is further described in detail below in conjunction with specific embodiments. The given examples are only for illustrating the present invention, but not for limiting the scope of the present invention.

[0054] The present invention is further described in detail below in conjunction with specific embodiments. The given examples are only for illustrating the present invention, but not for limiting the scope of the present invention.

[0055] The primer set for RAA amplification of Klebsiella pneumoniae nucleic acid provided by the present invention comprises an upstream primer and a downstream primer of a target double-stranded DNA; the nucleotide sequence of the upstream primer is shown in SEQ ID NO.:5; the nucleotide sequence of the downstream primer is shown in SEQ ID NO.:7.

[0056] The present invention designs DNA upstream primers and DNA downstream primers for the UreA gene in the genome of Klebsiella pneumoniae (hereinafter referred to as target double-stranded DNA), and uses them to perform room-temperature nucleic acid amplification on DNA extracted from a sample to be tested, and can obtain 10 within 10 to 30 minutes under a constant temperature of 25°C. 9 times the nucleic acid amplification product.

[0057] The Klebsiella pneumoniae detection composition provided by the present invention comprises:

[0058] An upstream primer and a downstream primer of the target double-stranded DNA, wherein the upstream primer comprises a T7 promoter;

[0059] A recombinase that can catalyze the formation of a double-stranded DNA containing a T7 promoter by an upstream primer and a first single-stranded target double-stranded DNA;

[0060] T7 RNA polymerase, which is used to catalyze the transcription of double-stranded DNA containing a T7 promoter to generate single-stranded RNA;

[0061] Transcriptase, which catalyzes the reverse transcription of single-stranded RNA into double-stranded DNA / RNA;

[0062] ATP energy regenerating enzyme, which is used to provide ATP energy in DNA recombination and transcription processes;

[0063] Helicase, which is used to catalyze the hydrolysis of DNA / RNA double strands into RNA single strands and DNA single strands;

[0064] Single-strand binding protein, which is used to bind to single-stranded DNA and prevent the self-polymerization of single-stranded DNA;

[0065] TaqMan fluorescent probe, which includes an RNA chain that can bind to a single-stranded DNA, and the 5' end and 3' end of the RNA chain have a fluorescent group and a quenching group respectively;

[0066] A cleavage enzyme, which is used to cleave the RNA chain of the TaqMan fluorescent probe to release the fluorescent group when the TaqMan fluorescent probe is bound to the DNA single chain;

[0067] in:

[0068] The nucleotide sequence of the upstream primer is shown in SEQ ID NO.: 5;

[0069] The nucleotide sequence of the downstream primer is shown in SEQ ID NO.: 7;

[0070] The nucleotide sequence of the TaqMan fluorescent probe is shown in SEQ ID NO.:15.

[0071] Simply put, the composition includes a nucleic acid amplification enzyme group and a signal amplification enzyme group. The nucleic acid amplification enzyme group contains 4 enzymes (recombinase, helicase, single-stranded binding protein, polymerase, ATP energy regeneration enzyme), and the signal amplification enzyme group contains 2 enzymes (transcriptase and cleavage enzyme). The present invention integrates the two groups of proteins together, so that enzyme-mediated room-temperature nucleic acid amplification (RAA for short) and signal amplification can be carried out in one reaction tube, simplifying the operation.

[0072] The present invention uses the above-mentioned Klebsiella pneumoniae detection composition to achieve 10 within 10 to 30 minutes under a constant temperature environment of 25°C. 9 The signal amplifier enzyme group can enable each nucleic acid amplification product to generate more than 10,000 fluorescent signals, significantly improving the speed and sensitivity of detection.

[0073] The TaqMan fluorescent probe used in the present invention is used to display the fluorescent signal generated during the target double-stranded DNA amplification process, and the fluorescent signal is proportional to the total amount of DNA bound to the TaqMan fluorescent probe. In some embodiments of the present invention, the quenching group of the TaqMan fluorescent probe is one of BHQ, BHQ1, BHQ2, and TAMRA, and the fluorescent group is one of FAM, FAM-X, CY3, CY5, HEX, and TET. Preferably, the 5' end of the TaqMan fluorescent probe is FAM and the 3' end is BHQ1.

[0074] In some embodiments, the Klebsiella pneumoniae detection composition further comprises a plasmid standard of the target double-stranded DNA for establishing a standard curve, or for calibrating and verifying the accuracy of quantitative detection of the target double-stranded DNA using the Klebsiella pneumoniae detection composition.

[0075] In some embodiments, the Klebsiella pneumoniae detection composition is used to amplify and detect target double-stranded DNA at 25°C without heating, is suitable for scenarios where heating is inconvenient, and has broad application prospects.

[0076] The Klebsiella pneumoniae detection kit provided by the present invention comprises the above-mentioned Klebsiella pneumoniae detection composition, which includes a primer pair and a TaqMan fluorescent probe for optimizing the RAA reaction. The use of the kit simplifies the detection process of Klebsiella pneumoniae and improves the sensitivity and specificity of the detection. Through the pre-configured reagent combination, the user can quickly start the Klebsiella pneumoniae detection without the need to prepare a reaction mixture separately, thereby reducing the complexity of the experimental operation and the potential risk of contamination.

[0077] In some embodiments, the Klebsiella pneumoniae detection kit provided by the present invention further comprises a lysate, which is composed of a broad-spectrum protease and a physiological buffer. The DNA extraction process can be completed by placing the sample in the lysate for a 10-minute heat treatment. The physiological buffer is preferably a Tris-EDTA buffer, and the pH value of the Tris-EDTA buffer is preferably 8.

[0078] In some embodiments, the Klebsiella pneumoniae detection kit provided by the present invention comprises a lyophilization protective agent, and each component in the Klebsiella pneumoniae detection composition is packaged in the form of a lyophilized powder, which can extend the shelf life of the kit, and the components can be mixed together when used. Alternatively, a system consisting of basic dry powder (recombinant enzyme, single-stranded binding protein, polymerase, ATP energy regeneration enzyme, signal amplification enzyme system), upstream primer, downstream primer, TaqMan fluorescent probe, activation solution NTP, buffer, RNase-free Water, and lyophilization protective agent is prepared into a pre-packed lyophilized tube.

[0079] When using this kit, follow the steps below:

[0080] S1. Collect samples, add lysis buffer, and incubate at 95°C for 10 minutes to obtain sample DNA;

[0081] S2. Add the sample DNA to the above kit for room temperature amplification reaction;

[0082] S3. The fluorescence signal Tt value is used as the Klebsiella pneumoniae detection result. If there is no Tt value in the cycle, it means that the sample to be tested is not infected with Klebsiella pneumoniae, and the test result is negative. If there is a Tt value, it means that the sample to be tested is infected with Klebsiella pneumoniae, and the test result is positive.

[0083] Optionally, step S2 is performed in a portable metal bath, and step S3 is detected using a portable fluorescence detector, and the detection is completed in 10 to 30 minutes. The kit of the present invention is used in conjunction with a portable metal bath and a portable fluorescence detector, and can be carried to any location such as a rural site for detection, meeting application scenarios such as environmental detection in experimental barriers and detection next to animal cages.

[0084] The Klebsiella pneumoniae detection device provided by the present invention comprises:

[0085] A DNA extraction module, which is used to extract sample DNA;

[0086] An amplification module, which is used to amplify the sample DNA using the Klebsiella pneumoniae detection composition to obtain an amplification product;

[0087] The fluorescence detection module is used to detect the fluorescence signal of the amplification product.

[0088] The detection device is designed to provide users with a complete solution from sample processing to result analysis, making the detection process of Klebsiella pneumoniae more automated and standardized, greatly improving laboratory work efficiency and the reliability of test results.

[0089] In some embodiments, the Klebsiella pneumoniae detection device provided by the present invention further includes a judgment module:

[0090] The judgment module judges whether the sample contains Klebsiella pneumoniae according to the detection result of the fluorescence detection module; if there is no fluorescence signal, it is judged that the sample does not contain Klebsiella pneumoniae; if there is a fluorescence signal, it is judged that the sample contains Klebsiella pneumoniae.

[0091] The technical solution of the present invention is described in detail below through specific embodiments:

[0092] Example 1

[0093] The Klebsiella pneumoniae positive samples used in the test were from Shanghai Chaorui Biotechnology Co., Ltd., trade name: Klebsiella pneumoniae fluorescent quantitative positive nucleic acid, PAER (DNA). The concentration was detected using a micro-spectrophotometer (Nanodrop) and then stored in a -80°C refrigerator.

[0094] Reaction system: Add 1 μL upstream primer (10 μM), 1 μL downstream primer (10 μM), 1 μL PCR product to an eight-tube tube containing basic dry powder (200 μL of 20-200 ng / μL helicase, 60 μL of 1-30 ng / μL recombinase, 240 μL of 100-1000 ng / μL single-stranded binding protein, 96 μL of 10-100 ng / μL polymerase, 80 μL of 1-10 ng / μL ATP energy regeneration enzyme, signal amplifier enzyme group including: 200 μL of 1-30 ng / μL DNA restriction endonuclease, 60 μL of 1-30 ng / μL auxiliary protein). TaqMan fluorescent probe (1μM), 7μL Klebsiella pneumoniae fluorescent quantitative positive nucleic acid, then centrifuged instantly, let stand at room temperature for 2 minutes, add 10μL activation solution (a solution composed of NTP (nucleoside triphosphate) and Tris-EDTA buffer) along the wall of the eight-tube, centrifuged instantly; shake and mix for 10s, centrifuge instantly and then test on the machine: portable PCR instrument, reaction temperature 25℃, signal collection once per cycle, 30 cycles in total, reaction time 30 minutes, the fluorescent group of TaqMan fluorescent probe is FAM, the quenching group is BHQ1, the fluorescent group is connected to the 5' end of RNA, and the quenching group is connected to the 3' end of RNA.

[0095] TaqMan fluorescent probe screening was performed using Klebsiella pneumoniae fluorescence quantitative positive nucleic acid as a template, Tris-EDTA buffer (hereinafter referred to as TE) as a negative control, and RNase-free Water (Takara, 9012) as a blank control.

[0096] The nucleotide sequences of the primers and probes designed by the present invention are shown in Table 1:

[0097] Table 1

[0098]

[0099]

[0100]

[0101] In Table 1, X represents the fluorescent group FAM.

[0102] Take the upstream primers F1, F5, F6, and the downstream primers R1, R5, R6 for cross-combination testing, namely F1R1, F5R1, F6R1, F1R5, F5R5, F6R5, F1R6, F5R6, F6R6, and add TaqMan fluorescent probes—RNA1, RNA2, RNA3, RNA4, RNA5, RNA6—to each primer group. Compare the corresponding identical primer groups and select the TaqMan fluorescent probe with the smallest Tt value and the highest endpoint fluorescence value. Then determine the corresponding 4 groups of DNA primer screening results based on the TaqMan fluorescent probe, and select the TaqMan fluorescent probe with the smallest Tt value and the highest endpoint fluorescence value, and the primer group with no amplification signal in the negative control.

[0103] The results showed that the above 9 primers F1R1, F4R1, F6R1, F1R4, F4R4, F6R4, F1R6, F4R6, F6R6 and 6 TaqMan fluorescent probes RNA1, RNA2, RNA3, RNA4, RNA5 and RNA6 were combined respectively, with a total of 54 combinations, 10 of which had good amplification effects. Among them, the Tt value of the F5R1RNA3 combination was the lowest at 5.5, and the fluorescence value was relatively high. There was no amplification signal in the negative control. It was determined that RNA3 was selected as the TaqMan fluorescent probe, and the F5 RNA3 combination was used for subsequent experiments ( Figure 2-Figure 5 ).

[0104] Example 2

[0105] Using Klebsiella pneumoniae fluorescence quantitative positive nucleic acid as a template, according to the reaction system of Example 1, RNA3 and upstream primer F5 were fixed, and downstream primers R1, R2, R3, R4, R5, and R6 were selected for screening, and the primer group with the smallest Tt value, the highest endpoint fluorescence value, and the negative control without amplification signal was selected. The results showed that in the screening results of the downstream primers, the Tt value of the F5R1 RNA3 group was low, the Tt value was 5.5, and the fluorescence value was relatively high, indicating that the downstream primer R1 had the best effect, the Tt value of the F5R5RNA3 group was the second lowest, the Tt value was 10.9, and R5 was the second, and there was no amplification signal in the negative control. Therefore, it was determined to select R1 as the downstream primer, and the combination of R1 RNA3 and R5 RNA3 was used to carry out subsequent experiments ( Figure 6 ).

[0106] Example 3

[0107] Using Klebsiella pneumoniae fluorescence quantitative positive nucleic acid as a template, according to the reaction system of Example 1, RNA3 and downstream primers R1 and R5 were fixed, and upstream primers F1, F2, F3, F4, F5, and F6 were selected for screening, and the primer group with the smallest Tt value, the highest endpoint fluorescence value, and no amplification signal in the negative control was selected. The results showed that in the screening results of the upstream primers, the Tt value of the F5R1 RNA3 group was relatively low, at 5.5, and the fluorescence value was relatively high; the Tt value of the F5R5 RNA3 group was 10.9. This shows that the upstream primer F5 has a better effect, and there is no amplification signal in the negative control. It is determined to select F5 as the upstream primer, and it is determined to use the combination of F5R1 RNA3 and F5R5 RNA3 to carry out the experiment ( Figure 7 ).

[0108] Example 4

[0109] Using Gram-negative bacteria (G - ): Commercial Klebsiella pneumoniae fluorescence quantitative positive nucleic acid, PAER (DNA), Commercial Salmonella fluorescence quantitative positive nucleic acid, PAER (DNA), Gram-positive bacteria (G + ): Commercial Klebsiella pneumoniae fluorescence quantitative positive nucleic acid, PAER (DNA), commercial Bacillus pilosus fluorescence quantitative positive nucleic acid, PAER (DNA) as template, according to the reaction system of Example 1, TE as negative control, RNase-free Water as blank control, and the optimal primer group F5R1 RNA3 obtained by screening was specifically detected according to the reaction system, time and temperature of Example 1. The results showed that two Gram-negative bacteria (G-) and two Gram-positive bacteria (G+) were selected for specificity experiments. The three tested bacteria were not detected, but Klebsiella pneumoniae was detected, indicating that the optimal primer group F5R1RNA3 screened by the present invention has high specificity ( Figure 7 ).

[0110] Example 5

[0111] This embodiment provides a method for detecting sample DNA:

[0112] Reaction system: A system containing basic dry powder (recombinant enzyme, single-stranded binding protein, polymerase, ATP energy regeneration enzyme, signal amplification enzyme system), upstream primer (F5), downstream primer (R1, R5), TaqMan fluorescent probe (RNA3), activation solution NTP (nucleoside triphosphate), buffer, RNase-free water, and lyophilization protective agent is prepared into eight-unit lyophilization tubes.

[0113] After adding 1 μL upstream primer (10 μM), 1 μL downstream primer (10 μM), 1 μL TaqMan fluorescent probe (1 μM), and 7 μL Klebsiella pneumoniae fluorescence quantitative positive nucleic acid to the eight-link freeze-dried tube, centrifuge instantly, let stand at room temperature for 2 minutes, add 10 μL activation solution (a solution composed of NTP (nucleoside triphosphate) and buffer) along the wall of the eight-link freeze-dried tube, centrifuge instantly; shake and mix for 10 seconds, centrifuge instantly, and then test on the machine. The detection instrument uses a portable PCR instrument, the reaction temperature is 25°C, and the signal is collected once per cycle, for a total of 30 cycles, the reaction time is 30 minutes, and the fluorescent group is set to FAM.

[0114] The number of commercial positive nucleic acid copies is 1×10 6 RNase-free water was used to perform gradient dilution of the plasmid standard containing the UreAgene gene fragment of Klebsiella pneumoniae, and the copy number was 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 , 1×10 2 , 10, 5, 1 were tested on the machine, and the optimal primer group F5R1RNA3 and F6R5 RNA3 were screened for sensitivity testing. The results showed that the primer probe screened by F5R1 RNA3 could detect a minimum copy number of 1, with high sensitivity, and the gradient diluted nucleic acid had a good effect on the machine ( Figure 8-9 ).

[0115] Example 6

[0116] Commercial Salmonella positive nucleic acid (hereinafter referred to as P1), commercial Corynebacterium muridarum positive nucleic acid (hereinafter referred to as P2) and Klebsiella pneumoniae fluorescent quantitative positive nucleic acid were used as positive controls, TE was used as a negative control, and RNase-free Water was used as a blank control. The optimal primer set F5R1RNA3 obtained by screening was used according to the reaction system, time, and temperature of Example 5 to determine the effect of the detection method of Example 5 in the sample. In addition, the same sample was taken for detection using the qPCR method. The results showed that the detection method of Example 5 was consistent with the detection results of the qPCR method, with an accuracy rate of 100% (Table 2 and Fig. 9 ).

[0117] Table 2 Comparison of the detection results of the detection method of Example 5 and the qPCR method

[0118]

[0119]

[0120] Example 7

[0121] The feces of five SPF-grade C57 mice were used as test samples. The test samples were added to the lysis solution and kept warm at 95°C for 10 minutes. The sample DNA was extracted to obtain samples A, B, C, D, and E. The Klebsiella pneumoniae fluorescent quantitative positive nucleic acid was used as a positive control, and TE was used as a negative control to determine the effectiveness of the detection method of Example 5 in on-site detection of actual samples. According to the detection method of Example 5, 5 samples were drawn in the laboratory and then tested on-site. The sample DNA was quickly extracted using the lysis solution, and the remaining samples were tested using the qPCR method. The results showed that the results of rapid on-site detection using the detection method of Example 5 were consistent with the results of the qPCR method, with an accuracy rate of 100% (Table 3 and Fig.10 ).

[0122] Table 3. Comparison of the detection results of the detection method of Example 5 and the qPCR method

[0123]

[0124] The foregoing is merely a specific embodiment of the present invention, which enables those skilled in the art to understand or implement the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A primer set for RAA amplification of Klebsiella pneumoniae nucleic acid, characterized in that: including an upstream primer and a downstream primer of a target double-stranded DNA; The nucleotide sequence of the upstream primer is shown in SEQ ID NO.: 5; The nucleotide sequence of the downstream primer is shown in SEQ ID NO.:

7.

2. A Klebsiella pneumoniae detection composition, characterized in that: The Klebsiella pneumoniae detection composition comprises: An upstream primer and a downstream primer of the target double-stranded DNA, wherein the upstream primer comprises a T7 promoter; A recombinase that can catalyze the formation of a double-stranded DNA containing a T7 promoter by an upstream primer and a first single-stranded target double-stranded DNA; T7 RNA polymerase, which is used to catalyze the transcription of double-stranded DNA containing a T7 promoter to generate single-stranded RNA; Transcriptase, which catalyzes the reverse transcription of single-stranded RNA into double-stranded DNA / RNA; ATP energy regenerating enzyme, which is used to provide ATP energy in DNA recombination and transcription processes; A helicase, which is used to catalyze the hydrolysis of the DNA / RNA double strand into RNA single strand and DNA single strand; Single-strand binding protein, which is used to bind to single-stranded DNA and prevent the self-polymerization of single-stranded DNA; TaqMan fluorescent probe, which includes an RNA chain that can bind to a single-stranded DNA, and the 5' end and 3' end of the RNA chain have a fluorescent group and a quenching group respectively; A cleavage enzyme, which is used to cleave the RNA chain of the TaqMan fluorescent probe to release the fluorescent group when the TaqMan fluorescent probe is bound to the DNA single chain; in: The nucleotide sequence of the upstream primer is shown in SEQ ID NO.: 5; The nucleotide sequence of the downstream primer is shown in SEQ ID NO.: 7; The nucleotide sequence of the TaqMan fluorescent probe is shown in SEQ ID NO.:

15.

3. The Klebsiella pneumoniae detection composition according to claim 2, characterized in that: The quenching group of the TaqMan fluorescent probe is one of BHQ, BHQ1, BHQ2, and TAMRA, and the fluorescent group is one of FAM, FAM-X, CY3, CY5, HEX, and TET.

4. The Klebsiella pneumoniae detection composition according to claim 2, characterized in that: The Klebsiella pneumoniae detection composition also includes a plasmid standard of the target double-stranded DNA.

5. The Klebsiella pneumoniae detection composition according to claim 2, characterized in that: The Klebsiella pneumoniae detection composition is used for amplifying and detecting target double-stranded DNA under a constant temperature condition of 25°C.

6. A Klebsiella pneumoniae detection kit, characterized in that: The Klebsiella pneumoniae detection kit comprises the Klebsiella pneumoniae detection composition according to any one of claims 2 to 5.

7. The Klebsiella pneumoniae detection kit according to claim 6, characterized in that: The Klebsiella pneumoniae detection kit further comprises a lysis solution, which comprises a broad-spectrum protease and a physiological buffer.

8. The Klebsiella pneumoniae detection kit according to claim 6 or 7, characterized in that: The Klebsiella pneumoniae detection kit comprises a lyophilization protective agent, and each component in the Klebsiella pneumoniae detection composition is packaged in the form of lyophilized powder.

9. A Klebsiella pneumoniae detection device, characterized in that: include: A DNA extraction module, which is used to extract sample DNA; An amplification module, which is used to perform RAA amplification on sample DNA using the Klebsiella pneumoniae detection composition according to any one of claims 2 to 5 to obtain an amplification product; The fluorescence detection module is used to detect the fluorescence signal of the amplification product.

10. The Klebsiella pneumoniae detection device according to claim 9, characterized in that: The Klebsiella pneumoniae detection device also includes a judgment module: The judgment module judges whether the sample contains Klebsiella pneumoniae according to the detection result of the fluorescence detection module; if there is no fluorescence signal, it is judged that the sample does not contain Klebsiella pneumoniae; if there is a fluorescence signal, it is judged that the sample contains Klebsiella pneumoniae.

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