Rapid antibacterial drug sensitivity test method

Abstract

The invention belongs to the technical field of microbiological detection, and particularly relates to a rapid antibacterial drug sensitivity test method. According to the method provided by the invention, the nucleic acid quantity of a specific gene fragment of bacteria is taken as a variable for monitoring phenotype AST, and the difference of nucleic acid copy numbers before and after antibiotic incubation is compared to reveal the sensitivity of the antibiotic. Specifically, aPCR and CRISPR-Cas12a are combined to construct a phenotype drug sensitivity detection system, a pair of universal primers in a 16S rDNA v3-v4 region is used for asymmetric nucleic acid amplification to obtain a large amount of ssDNA, then specific crRNA is designed to recognize a 16S rDNA specific fragment, Cas12a is activated to reversely cut F-QReporter to obtain fluorescence, and the purpose of signal detection is achieved. The phenotype drug sensitivity result is obtained within 4 hours according to the difference of the detection results before and after antibiotic incubation, and the method is efficient and reliable.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial detection, and specifically relates to a drug sensitivity detection method based on aPCR-CRISPR LbCas12a. Background Art

[0002] The emergence and spread of drug-resistant bacteria has become one of the most challenging public health issues today, posing a serious threat to human health and property. It is estimated that in 2019, there were about 1.27 million deaths caused by bacterial antimicrobial resistance. Antibiotic misuse and overuse are the main drivers of the development of drug-resistant pathogens. In hospitals, timely and accurate antimicrobial susceptibility test results can not only reduce the use of empirical broad-spectrum antibiotics and delay the development of drug-resistant bacteria, but also reduce the economic burden of expensive drugs on patients. However, the implementation of rapid AST (antimicrobial susceptibility test) platforms in clinical laboratories remains a major challenge.

[0003] At present, bacterial drug sensitivity detection methods are mainly divided into two categories: phenotypic AST and genotypic AST. As a drug sensitivity detection method based on bacterial culture, the results of traditional standard phenotypic AST methods and automated systems are accurate and reliable, but they have strict requirements on bacterial inoculation density and require at least 2-3 days to obtain drug sensitivity results. Genotype-based AST does not require bacterial enrichment and antibiotic culture, and can detect the presence of drug-resistant genes within a few hours. The advantages of sensitivity, specificity and high efficiency make this technology increasingly popular among researchers, such as RPA, FISH, qPCR, LAMP, etc. However, this method only detects drug-resistant genes with known drug resistance mechanisms, and drug-resistant genes are not completely consistent with phenotypic sensitivity. In recent years, researchers have developed new phenotypic drug sensitivity technologies that have greatly shortened the AST time. Phenotypic AST strategies are designed based on phenotypic characteristics such as bacterial turbidity, weight, metabolism, morphology and activity trajectory. The response of bacteria to antibiotics can be evaluated in a short time through absorbance, suspension arms, enzyme metabolism color development, real-time monitoring by microscope, Raman spectroscopy, etc. However, the above-mentioned drug sensitivity detection methods based on nonspecific characterization have high requirements on sample purity and are easily interfered by other commensal organisms. In general, they only reduce the drug sensitivity detection time but cannot avoid the time required for bacterial isolation and culture.

[0004] In summary, in order to detect infectious diseases and identify pathogens in the shortest possible time, as well as more importantly, drug sensitivity testing, an ideal rapid drug sensitivity testing system is required to have two key elements: 1. Good specificity to enable rapid identification of pathogens. 2. Not easily affected by commensal organisms, and can sensitively detect phenotypic changes of bacteria before and after short-term antibiotic incubation without the need to isolate single colonies and increase bacteria. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a rapid antimicrobial drug sensitivity test method.

[0006] The technical solution adopted by the present invention is as follows: a rapid antimicrobial drug sensitivity test method, the method is for non-disease diagnosis and treatment purposes, comprising the following steps:

[0007] (1) DNA extraction: extracting DNA from the sample to be tested;

[0008] (2) Asymmetric PCR amplification: asymmetric nucleic acid amplification of the DNA extracted in step (1) is performed using universal primers in the 16S rDNA v3-v4 region to obtain a PCR product;

[0009] (3) CRISPR LbCas12a detection: The CRISPR LbCas12a reaction system is co-incubated with the aPCR product obtained in step (2) to perform a CRISPR-Cas12a reaction. The CRISPR LbCas12a reaction system uses a specific crRNA that recognizes a specific fragment of 16S rDNA, and the fluorescence value result before antibiotic incubation is obtained by detection;

[0010] (4) Drug sensitivity test: The sample to be tested is added to a culture medium containing an antibiotic and incubated to obtain an antibiotic-incubated sample. Steps (1) to (3) are repeated for the sample after antibiotic incubation to obtain the fluorescence value after antibiotic incubation. The sensitivity of the pathogen to the antibiotic can be obtained by comparing the fluorescence values ​​obtained before and after antibiotic incubation.

[0011] Preferably, in step (1), DNA is extracted by repeated heating and freezing.

[0012] Preferably, in step (2), the primer ratio used in asymmetric PCR is 1:20 (Fw:Rv), and the final concentration of the limiting primer is 0.6 μM.

[0013] Preferably, in step (2), the asymmetric PCR conditions include: a pre-amplification step at 95°C for 3 minutes, followed by 35 cycles of denaturation at 95°C for 20 seconds, annealing at 59°C for 20 seconds, and extension at 72°C for 20 seconds. Finally, an extension step at 72°C for 3 minutes.

[0014] Preferably, in step (3), the CRISPR LbCas12a reaction system includes FQ Reporter and crRNA.

[0015] Preferably, in step (3), in the CRISPR LbCas12a reaction system, the final concentration of Cas12a is 100 nM, the final concentration of the FQ reporter probe is 300 nM, the final concentration of crRNA is 200 nM, and the incubation time of the CRISPR LbCas12a reaction system and the aPCR product is 20 minutes.

[0016] Preferably, in step (4), the sample to be tested is incubated with the culture medium containing antibiotics for 60 minutes.

[0017] Preferably, step (3) also includes a bacterial identification process, that is, targeting the specific crRNA of each bacterium to identify the bacteria, and the bacterial identification is completed.

[0018] The method provided by the present invention is to use the number of nucleic acids of bacterial specific gene fragments as the variable monitored by phenotypic AST, and the difference of nucleic acid copy number before and after antibiotic incubation is compared to reveal its sensitivity to antibiotics. Specifically, the present invention combines aPCR (asymmetric polymerase chain reaction) with CRISPR-Cas12a to construct a phenotypic drug sensitivity detection system, and asymmetric nucleic acid amplification is performed with a pair of universal primers in 16SrDNA v3-v4 district to obtain a large amount of ssDNA, and then specific crRNA is designed to recognize 16S rDNA specific fragments, and Cas12a is activated to trans-cut FQ Reporter to obtain fluorescence to achieve signal detection purposes, and the phenotypic drug sensitivity results are obtained within 4h according to the difference in the test results before and after antibiotic incubation, which is efficient and reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] 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 or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, without paying creative labor, other drawings obtained based on these drawings still belong to the scope of the present invention.

[0020] Figure 1 Schematic diagram of aPCR-CRISPR LbCas12a detection process;

[0021] Figure 2 Schematic diagram of aPCR-CRISPR LbCas12a detection system;

[0022] Figure 3: Preliminary feasibility RPA (a): Lanes 1-2 are negative, and Lane 3 is a positive control; PAM sequence (b) and non-PAM sequence products and sequence fragments (c), Lanes 1-6 correspond to: Escherichia coli (EC), Pseudomonas aeruginosa (PA), Klebsiella pneumoniae (KP), Enterococcus faecalis (EF), Staphylococcus aureus (SA);

[0023] Figure 4 Optimization of aPCR-CRISPR LbCas12a detection system conditions: annealing temperature (a), primer ratio (b), limiting primer concentration (c), PCR program (d) of aPCR, MHBⅡ volume (e), fluorescent probe concentration (f), crRNA concentration (g), and incubation time (h) of CRISPR-LbCas12a;

[0024] Figure 5 Performance evaluation: crRNA specificity (a), spiked urine detection (b), concentration gradient of six common urinary tract infection pathogens (ch);

[0025] Figure 6 Optimization of antibiotic incubation time;

[0026] Figure 7 rAST results of E. coli clinical isolates;

[0027] Figure 8 The aPCR-CRISPR LbCas12a detection system was used to identify pathogenic bacteria and perform drug sensitivity testing in clinical urine specimens. DETAILED DESCRIPTION

[0028] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings.

[0029] The present invention provides a drug sensitivity detection system based on aPCR-CRISPR LbCas12a, which is divided into two parts: bacterial identification and rapid drug sensitivity, wherein the rapid drug sensitivity part mainly includes the following steps:

[0030] (1) DNA extraction: extracting DNA from the sample to be tested;

[0031] (2) Asymmetric PCR amplification: Asymmetric nucleic acid amplification is performed on the DNA extracted in step (1) using universal primers in the 16S rDNA v3-v4 region (Table 1) to obtain a PCR product;

[0032] (3) CRISPR LbCas12a detection: The CRISPR LbCas12a reaction system was co-incubated with the aPCR product obtained in step (2) to perform a CRISPR-Cas12a reaction. The CRISPR LbCas12a reaction system used a specific crRNA that recognized a specific fragment of 16S rDNA (Tables 2 and 3), and the fluorescence value results before antibiotic incubation were obtained by detection;

[0033] (4) Drug sensitivity test: The sample to be tested is added to the culture medium containing antibiotics and incubated to obtain an antibiotic-incubated sample. Steps (1) to (3) are repeated for the sample after antibiotic incubation to obtain the fluorescence value after antibiotic incubation. The sensitivity of the pathogen to the antibiotic can be obtained by comparing the fluorescence values ​​obtained before and after antibiotic incubation. The ratio of the fluorescence signal before and after antibiotic incubation is called the fold change (FC), which is then compared with the threshold value to determine the sensitivity of Escherichia coli to the antibiotic.

[0034] The bacterial identification part is to identify the bacteria using specific crRNA targeting each bacterium after the universal 16S rDNA detection in step (3).

[0035] After the DNA in the sample to be tested is extracted, the overall process diagram is as follows: Figure 1 As shown:

[0036] 1. Start:

[0037] The process starts from "Start", and the first step is asymmetric PCR amplification to obtain aPCR product.

[0038] 2. Universal 16S rDNA detection (UNI crRNA):

[0039] Perform universal 16S rDNA test. If the result is "NO", it is judged as "No known infection" and the process ends. If the result is "Yes", proceed to the next step.

[0040] 3. Pathogen identification:

[0041] Perform pathogen identification to detect the presence of common pathogens, such as Escherichia coli (EC), Pseudomonas aeruginosa (PA), Klebsiella pneumoniae (PM), Streptococcus pneumoniae (KP), Enterococcus (EF), or Staphylococcus aureus (SA). If the result is "NO", it is determined to be "Other infection" and the process ends. If the result is "Yes", proceed to the next step.

[0042] 4. Antimicrobial Susceptibility Test (AST):

[0043] Perform an antimicrobial susceptibility test (AST). If the result of the AST shows "FC>threshold", the patient is considered "Susceptible". If the result of the AST shows "FC≤threshold", the patient is considered "Non-susceptible".

[0044] 5. Finish:

[0045] The process finally reaches "Finish", indicating that the entire inspection and testing process is completed.

[0046] The technical principle of the present invention is as follows Figure 2 As shown, the genome extracted from the test is amplified non-selectively and asymmetrically using a pair of universal primers in the 16S rDNA v3-v4 region: initially, the non-restrictive primer and the restrictive primer jointly participate in the symmetric amplification of the template to produce a large amount of double-stranded DNA (dsDNA). In the later stage of amplification, when the restrictive primer, i.e., the forward primer with a relatively low concentration, is consumed, the high-concentration reverse primer continues to use the double-stranded amplification product as a template to produce a large amount of target single-stranded DNA (ssDNA). In each CRISPR LbCas12a system, when crRNA recognizes the specimen, the initiated trans-cleavage protects the fluorescent group from the inhibition of the quenching group, and the fluorescence emitted, while the fluorescence intensity can reflect the concentration of the target bacteria: first, the universal crRNA (uni crRNA) is used to screen the amplified product to obtain a suspected bacterial infection specimen, and then the specific crRNA targeting each bacterium is used to identify the bacteria, so that the screening and identification of urine specimens positive for bacterial infection are completed.

[0047] Finally, the sample is further added to the culture solution containing antibiotics, where more 16S rDNA will be produced due to the reproduction of non-sensitive bacteria not affected by antibiotics, and sensitive bacteria are inhibited by antibiotics, and the total amount of 16S rDNA hardly increases. The sensitivity of pathogenic bacteria to antibiotics can be obtained by comparing the fluorescence values ​​obtained by target-guided LbCas12a trans-cutting before and after antibiotic incubation. Compared with conventional methods, the detection system of the present invention can complete bacterial identification and drug sensitivity detection within 5 hours, so it is faster and more convenient.

[0048] The following are some specific embodiments of the present invention, and the materials used are as follows:

[0049] Oligonucleotide sequence

[0050] Oligonucleotides were provided by Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China), and their sequences are shown in the table below.

[0051] Table 1 Universal primers for the 16S rDNA v3-v4 region in this experiment

[0052]

[0053] Table 2 crRNA transcription templates used in this experiment

[0054]

[0055] Table 3 PAM-free crRNA transcription templates used in this experiment

[0056]

[0057] Sample pretreatment

[0058] Clinical isolates: cryopreserved in 20% glycerol at -80°C. Before the experiment, the clinical isolates were inoculated into LB solid medium at 37°C overnight, and then single colonies were selected and enriched in MHBII (Mueller-Hinton II cation adjustment broth) (Chu Rui) for 12 hours. Subsequently, centrifugation was performed at 5000 rpm for 4 minutes at 4°C, the supernatant was discarded and sterile water was added to mix thoroughly, and the above operation was repeated for washing twice. The concentration was adjusted to OD600 = 1 using a spectrophotometer (Lingxi V-3000, Shanghai Lingxi Instrument Co., Ltd.), which is equivalent to about 10 9 cfu / mL concentration, the bacteria were diluted to different concentrations (10 3 -10 7 cfu / mL).

[0059] Urine specimen: According to the requirements of sterile procedures, the urine sample was centrifuged at 5000 rpm for 7 minutes at 4°C, the supernatant was discarded, and the same amount of MHBII as the supernatant was added, mixed thoroughly, and divided into 2 portions, and then stored at -80°C.

[0060] After optimization, the specific process of each step of the present invention is as follows:

[0061] (1) DNA extraction:

[0062] The prepared bacterial suspension was repeatedly heated and frozen to obtain the bacterial genome: 100°C for 2 minutes and 4°C for 1 minute, repeated 3 times, and then stored at -80°C. Before amplification, it was re-dissolved and centrifuged at 10,000 rpm for 1 minute at 4°C, and the supernatant was taken for subsequent amplification.

[0063] (2) Asymmetric PCR amplification:

[0064] The primers used in the aPCR assay were obtained from previous studies. The primer ratio used for asymmetric PCR was 1:20 (Fw:Rv), the final concentration of the limiting primer was 0.6 μM in a 25 μL reaction mixture, and the remaining reagents were added according to the instructions of the Katara ExTaq (R001A) kit. The PCR conditions used included a preamplification step at 95°C for 3 minutes, followed by 35 cycles of denaturation at 95°C for 20 seconds, annealing at 59°C for 20 seconds, and then extension at 72°C for 20 seconds. Finally, an extension step was performed at 72°C for 3 minutes. The amplification product was confirmed by agarose gel electrophoresis.

[0065] (3) CRISPR LbCas12a:

[0066] In a 20 μL system, 9 μL H2O, 2 μL NE buffer (New Egland Biolabs), 2 μL 2 μM crRNA, 3 μL 2 μM FQ reporter probe, 2 μL 1 μM Cas12a (New Egland Biolabs), and 2 μL aPCR product were added. Gently mix and incubate at 37°C for 20 minutes, then immediately placed in a 4°C 384-well plate to determine the fluorescence intensity at 520 nM.

[0067] (4) Antibiotic incubation

[0068] Ciprofloxacin hydrochloride monohydrate and gentamicin sulfate were purchased from Aladdin (Shanghai, China). These antibiotics were dissolved in H2O and aliquoted into 25 μg / mL ciprofloxacin and stored at -20°C, and gentamicin was diluted to 400 μg / mL and stored at 4°C. Each aliquot of antibiotics was used only once and did not need to be refrozen. 495 μL of 10 5 The MHBII suspension of 1 cfu / mL bacteria was mixed with 5 μL of diluted antibiotics to make a final CIP concentration of 0.25 μg / mL and a final GM concentration of 4 μg / mL and placed in a 37 °C incubator for 60 min before DNA extraction immediately.

[0069] Embodiment 1:

[0070] Taking Escherichia coli (containing PAM) and Klebsiella pneumoniae (without PAM) as examples, aPCR was selected as the amplification tool to produce a large amount of ssDNA, such as Figure 3 c. The annealing temperature, primer ratio, primer concentration and PCR program of asymmetric amplification were optimized, as shown in Figure 4As shown in c, when the final concentration of the non-restrictive primer was 0.6 micromolar, 1:20 showed the highest signal intensity. By optimizing the PCR program, asymmetric amplification can be completed in only 62 minutes. Finally, the bacteria were dissolved in MHBⅡ and lysed at high temperature. Figure 4 eThe results showed that the best amplification effect was achieved when 2 μL of lysis buffer was added to the amplification system, and the salt ion concentration in the excessive MHBⅡ would reduce the amplification efficiency.

[0071] In order to improve the detection performance, the output fluorescence signal can clearly distinguish the changes in bacterial concentration. Taking Escherichia coli (containing PAM) and Klebsiella pneumoniae (without PAM) as examples, two concentrations (10 4 cfu / mL / 10 6 cfu / mL) to optimize the FQ Reporter, crRNA, incubation time and MHBⅡ addition amount of the CRISPR LbCas12a cutting system. Figure 4 As shown in Figure 5, in this example, the final concentration of Cas12a is fixed at 100 nM. As FQ increases, when the final concentration of FQ is 300 nM, the signal difference between the two concentrations increases significantly. In the concentration range of 100-400 nM, Figure 4 g, the fluorescence signal increases with the crRNA concentration, but considering that too high a signal is not conducive to distinguishing between high-concentration bacteria, 200nM was selected as the optimal crRNA concentration. Finally, it was found that only 20 minutes of cutting incubation time was needed to obtain the best signal difference. Figure 4 h.

[0072] Comparative Example 1:

[0073] In step (2), RPA (recombinase polymerase amplification) is selected as the amplification tool, which is easy to cause false positives in the detection of UPEC (urinary tract infection pathogenic Escherichia coli), such as Figure 3 As shown in a.

[0074] Embodiment 2:

[0075] Good specificity is not only the basic prerequisite for accurate detection, but also one of the key factors in achieving subsequent rapid drug sensitivity.

[0076] For 6 common urinary tract infection pathogens (Escherichia coli, Bacillus, Pneumoniae, Escherichia coli, Aeruginosa, Staphylococcus aureus), ssDNA was selected as the target. The crRNA designed in this way has no PAM restriction, is more convenient and flexible, and multiple base differences can achieve the purpose of specific detection. Figure 5As shown in a, the specific crRNA of each bacteria designed can clearly distinguish the target bacteria from other bacteria, and the fluorescence signal is significantly higher than that of other bacteria. At the same time, it can also specifically identify the target in mixed bacterial detection. These results show that the crRNA designed by this method has the advantages of good specificity and strong anti-interference ability.

[0077] The 10 3 -10 7 Fluorescence signal results in the cfu / mL range Figure 5 Here, LOD is defined as three times the standard deviation of the blank measurement (S / N=3), which shows that the sensitivity of this method reaches 10 3 cfu / mL, the fluorescence signal increased with the increase of bacterial concentration, among which 10 3 and 10 4 The signal difference between cfu / mL is small, with a value of 10 4 -10 6 The increase in cfu / mL is obvious, which clearly demonstrates the potential of the method of the present invention in subsequent drug sensitivity testing applications. In order to apply the method to actual samples in the future, this example further optimizes the urine sample processing process, centrifuges the urine sample under low temperature conditions, and replaces the supernatant with MHBⅡ. According to the pretreatment process, this example adds a known concentration of standard Escherichia coli (10 4 , 10 6 cfu / mL) were added to normal urine, proteinuria and hematuria for detection, such as Figure 5 As shown in b, pretreatment had almost no effect on bacterial concentration and maintained bacterial activity significantly longer than that of untreated specimens, indicating that the proposed method was accurate.

[0078] Comparative Example 2:

[0079] For 6 common urinary tract infection pathogens (Escherichia coli, Bacillus thuringiensis, Pneumoniae, Escherichia coli, Aeruginosa, Staphylococcus aureus), the fragments containing PAM sequences in the product fragments were selected as targets, and each bacteria was injected into the culture medium at a high concentration (10 7 cfu / mL) as the detection object, it was found that the crRNA specificity of Proteus mirabilis and Klebsiella pneumoniae was not ideal, and there was mutual interference. This was because the base similarity of the two targets in the near PAM region was extremely high, and the difference between the two targets occurred at the far PAM end, resulting in a decrease in the recognition efficiency of the CRISPR system. Figure 3 As shown in b, it is difficult to find targets with large differences in bases near the PAM end of the two bacteria in this product fragment.

[0080] Embodiment 3:

[0081] Taking Escherichia coli as the detection object, we selected two commonly used antibiotics, ciprofloxacin and gentamicin, which belong to fluoroquinolones and aminoglycosides respectively. According to the Escherichia coli standard of the Clinical Laboratory Standards Institute (CLSI (M100)), the concentration of ciprofloxacin was 0.25 μg / mL and the concentration of gentamicin was 4 μg / mL (MIC-S). In the aPCR LbCas12a detection system, the fluorescence value when the antibiotic incubation time was 0 minutes was recorded as F0, the fluorescence value after the antibiotic incubation for a period of time was recorded as F1, and the ratio of the two was recorded as FC. The results are shown in Figure 6 It shows that compared with the blank group, the growth of sensitive Escherichia coli was significantly inhibited after 60 minutes of antibiotic incubation, while the growth of non-sensitive Escherichia coli was not inhibited and was the same as the blank group. At this time, the FC values ​​of sensitive Escherichia coli and non-sensitive Escherichia coli were significantly different, so 60 minutes was selected as the antibiotic incubation time. Compared with the antibiotic incubation time of 16 hours for conventional AST, the method of the present invention is faster, more sensitive and more convenient.

[0082] Embodiment 4:

[0083] In order to verify the feasibility and accuracy of this method in AST (antimicrobial susceptibility test), this example uses 17 clinical UPEC urinary tract infection specimens as the detection object. According to the optimized conditions, after 60 minutes of antibiotic incubation and aPCR-CRISPR LbCas12a detection, the results are as follows Figure 7 As shown in the figure, there was a very significant difference in FC values ​​between the sensitive and non-susceptible isolates to the two antibiotics (p<0.01). These results were consistent with those of conventional AST, so this method was accurate for AST of clinical isolates.

[0084] Embodiment 5:

[0085] In order to further explore the feasibility of this method in detecting real samples, this example takes uncomplicated urinary tract infection as the main research object, and collects a total of 61 clinical suspected urinary tract infection samples. Figure 1 The test process shown in the figure follows the diagnostic process of the clinical laboratory center for each urine specimen: including bacterial enrichment and purification, identification of pathogenic bacteria by Bruker mass spectrometer, and microbroth dilution as the gold standard for drug sensitivity test results. The clinical ID / AST report for each specimen takes at least 48 hours.

[0086] Using the aPCR-CRISPR LbCas12a detection system, refrigerated urine specimens can be rapidly screened (100 minutes), identified as bacterial species (30 minutes), and rapidly tested for drug sensitivity (120 minutes) in a short period of time. Among them, 41 specimens of urinary tract infection were found to have bacterial infection, and the results were basically consistent with the clinical results based on culture, 95.12%. ( Figure 8 a) Two samples were negative, but culture indicated Candida albicans infection. This is because the universal crRNA designed by the present invention mainly identifies common urinary tract infection pathogens, and cannot identify uncommon urinary tract infection pathogens such as fungi. 4 cfu / mL, Gram-negative bacteria concentration is >~10 5 cfu / mL, as the standard for judging urinary tract infection, the specific crRNA designed by the method provided by the present invention successfully identified 6 common urinary tract infection pathogens in 29 specimens and obtained the relative concentration of infected bacteria, and the results were consistent with the clinical data, 100%. ( Figure 8 b)

[0087] According to the identification results, 18 UPEC-infected urine samples were required to undergo drug sensitivity testing. The bacterial concentration in the samples was diluted to 10 5 cfu / mL, according to the optimized process, the AST results of this method are 36 data, the test results are as follows Figure 8 c, d, the receiver operating curve analysis (ROC) generated calculates the maximum Youden index as the threshold value of CIP and GM sensitivity and insensitivity, at this time, sensitive samples and insensitive samples can be clearly distinguished, where the point falls above the threshold line to indicate insensitive samples, and the point falls below the threshold line to indicate sensitive samples. The yellow point represents that the result is inconsistent with the standard method result, wherein the sensitive sample is 100% (13 / 13) and the insensitive sample is 95.65% (22 / 23). The observation area under the generated ROC curve (AUC) CIP is 0.95, and GM is 0.96. The results show that the method of the present invention is suitable for directly using urine instead of a single colony as the starting material for AST. However, due to the limited conditions and the number of clinical urine specimens collected, this embodiment only selects Escherichia coli for rapid drug sensitivity detection of commonly used antibiotics in fluoroquinolones and aminoglycosides, both of which play a bactericidal role by hindering DNA synthesis and interfering with protein synthesis, while other types of antibiotics such as β-lactams that inhibit cell wall synthesis have not been verified. Fortunately, the results of validated antibiotic susceptibility testing are very accurate, so the performance of this method meets the requirements for the identification and susceptibility testing of pathogens of simple urinary tract infections.

[0088] The above disclosure is only the preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. A rapid antimicrobial susceptibility test method, which is for purposes other than disease diagnosis and treatment, characterized in that: The following steps are involved: (1) DNA extraction: extracting DNA from the sample to be tested; (2) Asymmetric PCR amplification: asymmetric nucleic acid amplification of the DNA extracted in step (1) is performed using universal primers in the 16S rDNA v3-v4 region to obtain a PCR product; (3) CRISPR LbCas12a detection: The CRISPR LbCas12a reaction system is co-incubated with the aPCR product obtained in step (2) to perform a CRISPR-Cas12a reaction. The CRISPR LbCas12a reaction system uses a specific crRNA that recognizes a specific fragment of 16S rDNA, and the fluorescence value result before antibiotic incubation is obtained by detection; (4) Drug sensitivity test: The sample to be tested is added to a culture medium containing an antibiotic and incubated to obtain an antibiotic-incubated sample. Steps (1) to (3) are repeated for the sample after antibiotic incubation to obtain the fluorescence value after antibiotic incubation. The sensitivity of the pathogen to the antibiotic can be obtained by comparing the fluorescence values ​​obtained before and after antibiotic incubation.

2. The rapid antimicrobial susceptibility test method according to claim 1, characterized in that: In step (1), DNA is extracted by repeated heating and freezing.

3. The rapid antimicrobial susceptibility test method according to claim 1, characterized in that: In step (2), the primer ratio used in asymmetric PCR was 1:20 (Fw:Rv), and the final concentration of the limiting primer was 0.6 μM.

4. The rapid antimicrobial drug sensitivity test method according to claim 3, characterized in that: In step (2), the asymmetric PCR conditions included a preamplification step at 95°C for 3 minutes, followed by 35 cycles of denaturation at 95°C for 20 seconds, annealing at 59°C for 20 seconds, and extension at 72°C for 20 seconds. Finally, an extension step was performed at 72°C for 3 minutes.

5. The rapid antimicrobial drug sensitivity test method according to claim 1, characterized in that: In step (3), the CRISPR LbCas12a reaction system includes FQ Reporter and crRNA.

6. The rapid antimicrobial drug sensitivity test method according to claim 5, characterized in that: In step (3), in the CRISPR LbCas12a reaction system, the final concentration of Cas12a is 100nM, the final concentration of the FQ reporter probe is 300nM, the final concentration of crRNA is 200nM, and the incubation time of the CRISPR LbCas12a reaction system and the aPCR product is 20 minutes.

7. The rapid antimicrobial susceptibility test method according to claim 1, characterized in that: In step (4), the sample to be tested is incubated with the culture medium containing antibiotics for 60 minutes.

8. The rapid antimicrobial drug sensitivity test method according to claim 1, characterized in that: Step (3) also includes a bacterial identification process, that is, targeting the specific crRNA of each bacterium to identify the bacteria, and the bacterial identification is completed.

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