A multiple nucleic acid detection method based on loop-mediated isothermal amplification technology and nuclease

Abstract

The present application relates to the technical field of gene detection, and in particular to a multiple nucleic acid detection method based on loop-mediated isothermal amplification technology and nuclease, which comprises the following steps: designing primers for targets, amplifying the sample to be tested by using the loop-mediated isothermal amplification technology based on the primers to obtain an amplification product; and digesting the amplification product by using a heat-sensitive double-strand specific enzyme and performing fluorescence detection. The method combines LAMP and ezDNase, which not only shortens the detection time and reduces the detection cost, but also has high sensitivity, strong specificity and can perform multiple detection.

Description

Technical Field

[0001] This invention relates to the field of gene detection technology, and in particular to a multiplex nucleic acid detection method based on loop-mediated isothermal amplification technology and nucleases. Background Technology

[0002] In situations with limited resources, rapid and accurate on-site detection of pathogen DNA is crucial for the prevention, control, and timely treatment of infectious diseases. However, traditional pathogen detection methods require stringent laboratory conditions, making them unsuitable for on-site testing. While isothermal amplification and nuclease-based diagnostic methods have made significant progress in point-of-care testing (POCT), they still face numerous problems and challenges in practical applications. For example, effective multiplex nucleic acid detection has been difficult to achieve due to the different substrates targeted by Cas proteins and their indiscriminate trans-cleavage. Furthermore, the identification of the guide sequence takes time, slowing down the detection process. Moreover, existing nucleic acid detection technologies suffer from high costs and require expensive and complex instruments, hindering widespread application in resource-scarce areas such as point-of-care testing, primary hospitals, and remote regions. LAMP technology is inexpensive and easy to operate, requiring only a metal bath or water bath for the reaction. However, LAMP technology relies on a single tube and single reaction, failing to provide reliable multiplex detection signals, severely impeding its application.

[0003] Therefore, developing new diagnostic methods that are easy to implement, highly sensitive, flexible, and do not affect the accuracy of diagnostic results is of great significance for the prevention and control of infectious diseases in resource-limited areas. Summary of the Invention

[0004] The purpose of this invention is to provide a multiplex nucleic acid detection method based on loop-mediated isothermal amplification technology and nuclease, which has the advantages of short detection time, simplicity, high sensitivity, and strong multi-channel detection capability.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a multiplex nucleic acid detection method based on loop-mediated isothermal amplification technology and nucleases, comprising the following steps: (1) Primers were designed for the target, and loop-mediated isothermal amplification technology was used to amplify the sample to be tested based on the primers to obtain the amplification product; (2) The amplified product was digested with a thermosensitive double-stranded specific enzyme and then detected by fluorescence.

[0006] Preferably, in step (1), the primers include inner primers, outer primers, and loop primers; The inner primers are modified with fluorescent groups and quenching groups.

[0007] Preferably, the fluorescent group includes FAM, VIC, and CY5; the quenching group includes BHQ1 and BHQ2.

[0008] Preferably, in step (1), the amplification reaction system consists of 0.6-1.0 μL primer mixture, 1.4-1.8 μL dNTPs, 3.0-3.4 μL betaine, 1.4-1.8 μL template, 1.8-2.2 μL 10×Bst buffer, 0.6-1.0 μL Bst polymerase, and 8.8-11.2 μL water.

[0009] Preferably, the primer mixture, using nuclease-free ultrapure water as a solvent, comprises the following components at final concentrations: 1.4–1.8 μM inner primer, 0.1–0.3 μM outer primer, and 0.2–0.6 μM loop primer; The concentration of the dNTPs is 8-12 nM; the concentration of the betaine is 8-12 nM; and the concentration of the Bst polymerase is 6-10 U / μL.

[0010] Preferably, in step (1), the amplification reaction procedure is: incubation at 60~70℃ for 15~25 min.

[0011] Preferably, in step (2), the digestion reaction system is: 8~12μL amplification product, 0.8~1.2μL thermosensitive double-stranded specific enzyme, 1.8~2.2μL 10×dsDNase buffer, and 4.6~9.4μL water.

[0012] Preferably, the concentration of the thermosensitive double-stranded specific enzyme is 1~5 U / μL.

[0013] Preferably, in step (2), the digestion reaction procedure is: incubation at 25~40℃ for 5~20 min.

[0014] Beneficial effects: This invention provides a multiplex nucleic acid detection method based on loop-mediated isothermal amplification (LAMP) and nucleases. It combines LAMP with a thermosensitive double-stranded DNA-specific enzyme (ezDNase) for detection. This method features a robust and direct reaction system and workflow. The multiplex nucleic acid detection method only requires modifying the isothermal amplification primers with fluorescence resonance energy transfer (FRET) groups. Relying on the double-stranded DNA (dsDNA) recognition characteristics and non-specific cleavage mechanism of ezDNase, the entire detection process can be completed without additional reaction components. It provides simple, stable, and easily stored reaction reagents; the entire reaction can be completed within 30 minutes. The sensitivity of multiplex nucleic acid detection methods is between 1 and 10 copies / μL; the sensitivity of multiplex nucleic acids to real samples is 93.9% to 100%. The multiplex nucleic acid detection method of the present invention has the advantages of short testing time, simplicity, high sensitivity, and strong multi-channel detection capability; these outstanding features make it a powerful tool for detecting the nucleic acid of infectious pathogens and identifying their subtypes. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of a multiplex nucleic acid detection method based on loop-mediated isothermal amplification technology and nucleases, where a is a schematic diagram of the working principle of the detection method, b is a schematic diagram of the non-specific cleavage mechanism of ezDNase, and c is a schematic diagram of multicolor fluorescence signal output. Figure 2 The results show the feasibility verification of a multiplex nucleic acid detection method based on loop-mediated isothermal amplification technology and nucleases. In the figure, a is a schematic diagram of ezDNase recognition and cleavage of LAMP amplification products, b is the result of verification of the cleavage effect of ezDNase on LAMP amplification products by native-PAGE, c is the result of ultraviolet irradiation, d is the result of fluorescence detection, e is a schematic diagram of the adsorption of digestion products on amino silica microspheres, and f is the microscopic observation result of fluorescently labeled microspheres. Figure 3 The results show the performance of the multiplex nucleic acid detection method, where a is a schematic diagram of the multiplex nucleic acid detection method, b is the detection time of the multiplex nucleic acid detection method, c is the sensitivity measurement result of the multiplex nucleic acid detection method, d is a schematic diagram of multiplex nucleic acid multipath detection, e is the visualization result of the multiplex nucleic acid detection method for identifying HPV subtypes; and f is the microscopic observation result of fluorescently labeled microspheres. Figure 4 To obtain specific results for detecting Pseudomonas aeruginosa using multiplex nucleic acid detection methods; Figure 5 To assess the sensitivity and specificity of multiplex nucleic acid detection for Vibrio parahaemolyticus, where a represents the sensitivity result and b represents the specificity result; Figure 6 To demonstrate the sensitivity and specificity of multiplex nucleic acid detection for EBV virus, where a represents the sensitivity result and b represents the specificity result; Figure 7 The fluorescence amplification curves for identifying HPV subtypes using multiplex nucleic acid detection methods are shown, where 1 is the negative control, 2-4 are single-target detection results, 5-7 are dual-target detection results, and 8 is a triple-target detection result. Figure 8 The results are for clinical samples, where a) are multiplex nucleic acid test results of 79 Pseudomonas aeruginosa samples, b) are multiplex nucleic acid test results of 21 Vibrio parahaemolyticus samples, and c) are multiplex nucleic acid test results of 51 EBV samples. Figure 9The results are from HPV clinical sample testing, where a represents the results of multiplex nucleic acid testing, and b represents a comparative analysis of the results of multiplex nucleic acid testing and qPCR testing. Detailed Implementation

[0016] This invention provides a multiplex nucleic acid detection method based on loop-mediated isothermal amplification technology and nucleases, comprising the following steps: (1) Primers were designed for the target, and loop-mediated isothermal amplification technology was used to amplify the sample to be tested based on the primers to obtain the amplification product; (2) The amplified product was digested with a thermosensitive double-stranded specific enzyme and then detected by fluorescence.

[0017] In this invention, in step (1), the primers include inner primers, outer primers, and loop primers; The inner primers are modified with fluorescent groups and quenching groups; The fluorescent group is attached to the 5' end of the inner primer, and the quenching group is modified on the T base of the inner primer.

[0018] In this invention, the fluorescent groups include FAM, VIC, and CY5; the quenching groups include BHQ1 and BHQ2.

[0019] In this invention, before the sample is tested, the nucleic acid extraction process is complex and time-consuming, which will significantly prolong the overall analysis time. This situation is not suitable for the POCT testing environment for infectious diseases. Therefore, in the sample processing, nucleic acid extraction can be omitted, and nucleic acid lysis buffer can be used. The lysis process only takes 1 to 3 minutes to complete.

[0020] In this invention, in step (1), the amplification reaction system is: The primer mixture is 0.6–1.0 μL, preferably 0.7–0.9 μL, and more preferably 0.8 μL. The concentration of dNTPs is 1.4~1.8 μL, preferably 1.5~1.7 μL, and more preferably 1.6 μL. The amount of betaine is 3.0~3.4 μL, preferably 3.1~3.3 μL, and more preferably 3.2 μL. The template volume is 1.4~1.8 μL, preferably 1.5~1.7 μL, and more preferably 1.6 μL. The 10×Bst buffer is 1.8~2.2μL, preferably 1.9~2.1μL, and more preferably 2.0μL. Bst polymerase: 0.6–1.0 μL, preferably 0.7–0.9 μL, more preferably 0.8 μL. The water concentration is 8.8~11.2 μL, preferably 9.4~10.6 μL, and more preferably 10 μL.

[0021] In this invention, the primer mixture, using nuclease-free ultrapure water as a solvent, comprises the following components at the following final concentrations: The inner primer is 1.4–1.8 μM, preferably 1.5–1.7 μM, and more preferably 1.6 μM. The outer primer is 0.1~0.3μM, preferably 0.2μM. The circular primer has a concentration of 0.2–0.6 μM, preferably 0.3–0.5 μM, and more preferably 0.4 μM; The concentration of the dNTPs is 8-12 nM, preferably 9-11 nM, and more preferably 10 nM; the concentration of the betaine is 8-12 nM, preferably 9-11 nM, and more preferably 10 nM; the concentration of the Bst polymerase is 6-10 U / μL, preferably 7-9 U / μL, and more preferably 8 U / μL.

[0022] In this invention, in step (1), the amplification reaction procedure is: incubation at 60~70℃ for 15~25 min; The incubation temperature is preferably 63~67℃, and more preferably 65℃; The incubation time is preferably 18-22 minutes, and more preferably 20 minutes.

[0023] In this invention, the digestion reaction system in step (2) is: The amplification product is 8-12 μL, preferably 9-11 μL, and more preferably 10 μL. The thermosensitive double-stranded specific enzyme is administered at a concentration of 0.8–1.2 μL, preferably 0.9–1.1 μL, and more preferably 1.0 μL. The 10×dsDNase buffer is 1.8~2.2μL, preferably 1.9~2.1μL, and more preferably 2.0μL. The water concentration is 4.6 to 9.4 μL, preferably 5.8 to 8.2 μL, and more preferably 7.0 μL.

[0024] In this invention, the concentration of the thermosensitive double-stranded specific enzyme is 1~5 U / μL, preferably 2~4 U / μL, and more preferably 3 U / μL.

[0025] In this invention, the digestion reaction procedure in step (2) is: incubation at 25~40℃ for 5~20 min; The incubation temperature is preferably 30~35℃, more preferably 32℃; The incubation time is preferably 10-15 minutes, and more preferably 12 minutes.

[0026] In this invention, the multiplex nucleic acid detection method (working principle as follows) Figure 1 a) in this context refers to rapid nucleic acid amplification using LAMP, which offers excellent sensitivity. The 5' end of the internal primer is then modified with a fluorescent group, and a quenching group is added to the T base within the effective quenching range of FAM to form the internal primer molecular probe. ezDNase is specific for dsDNA; under appropriate conditions, it can recognize and digest dsDNA, almost ignoring the presence of single-stranded DNA (ssDNA). Figure 1 (b) The inner primer probe remains intact in the ezDNase digestion environment, and the detection of the fluorescence signal produces almost no background noise. During nucleic acid amplification, the inner primer probe binds to the target to form dsDNA, achieving high specificity through base sequence complementarity. The dsDNA amplification product rapidly activates a large amount of ezDNase in the reaction solution, simultaneously inducing its cleavage activity to digest the amplification product. During this process, the inner primer probe is also rapidly cleaved, thereby generating a detectable fluorescence signal. Although the inner primer probe is located at one end of the amplification product, this does not reduce its consumption rate because its length is sufficient for cleavage. Traditional CRISPR nucleic acid detection methods rely on a trans-cleavage mechanism, which differs from multi-channel detection methods based on multicolor fluorescence channels. The detection methods are incompatible; however, this invention suggests that non-specific cleavage mechanisms can still be used to design multi-target detection strategies, provided a specific system is used to guide selective cleavage. The ezDNase-mediated dsDNA recognition properties can be used to guide the selective operation of its indiscriminate cleavage mechanism. Simultaneously, based on the sequence specificity of the inner primer probe, multiplex nucleic acid detection methods achieve bispecificity for both nucleotide sequences and double-stranded DNA. When targeting nucleic acids from various pathogens, LAMP primers bind to the target and amplify it into dsDNA. Upon ezDNase activation, the dsDNA amplification product is selectively and non-specifically cleaved (all amplification products are cleaved regardless of whether a single or multiple targets are present in the system), generating corresponding multicolor fluorescent signals. Figure 1 (c in the text)

[0027] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0028] In this invention, all oligonucleotides, modified primers, gRNAs, and plasmids were synthesized by Bioligo Biotechnology (Shanghai) Co., Ltd.; the modified primers were internal primer molecular probes with fluorescent groups attached to the 5' end of the sequence and quenching groups modified on the T bases of the sequence; the plasmid vector carrying the target gene was pUC57. ezDNase protein was purchased from Harbin Xinhai Gene Testing Co., Ltd., China, catalog number: C5010; LAMP amplification kit and sample lysis buffer were purchased from Wuhan HanHai New Enzyme Biotechnology Co., Ltd. (catalog numbers: HMD5213-02 and HMD3504-02); UltraStartSYBR Green qPCR Master Mix was purchased from Chengdu Rongwei Gene Biotechnology Co., Ltd. (catalog number: A403). Extraction of bacterial genome: Genomic DNA was extracted from cultured Pseudomonas aeruginosa and Vibrio parahaemolyticus specimens according to the instructions provided with the bacterial genomic DNA extraction kit; the concentration of the extracted nucleic acid was then determined using a multi-mode microplate reader and diluted with ultrapure water to 1.0 × 10⁻⁶. 6 Copy / μL, store at -20℃ for later use.

[0029] Bacterial DNA extraction kits were purchased from Hunan Aikerui Biotechnology Co., Ltd. (catalog number: AG21007); amino silica microspheres were purchased from Nanjing Jike Biotechnology Co., Ltd. (catalog number: JK-04-003-5000); 1% agarose pre-prepared gels were purchased from Guangzhou Baiguang Biotechnology Co., Ltd. (catalog number: APG2001); DNA markers were provided by Beijing Lanbolide Trading Co., Ltd. (catalog number: L0500); LB, TSA, and TSB culture media were purchased from Beijing Sora Biotechnology Co., Ltd. (catalog numbers: L1010, T8650, LA0110); NaCl solution, Tris-HCl solution, nuclease inhibitors, enzyme-free water, and DEPC-treated water were all purchased from Sangon Biotech (Shanghai) Co., Ltd. Culture media and agar plates were provided by the Microbiology Laboratory of the Joint Laboratory of Hainan Medical University; Pseudomonas aeruginosa was inoculated on LB solid plates, and Vibrio parahaemolyticus was inoculated on TSA (containing 3% NaCl); after incubation at 37°C, single colonies were picked and incubated overnight at 37°C in 5 mL LB and TSB (containing 3% NaCl) media, respectively.

[0030] Example 1 Feasibility Verification

[0031] LAMP technology possesses excellent sensitivity and specificity, and reagents are readily available at low cost. Therefore, this invention combines LAMP technology with ezDNase to establish a multiplex nucleic acid detection method: (1) Primers were designed for the target of Pseudomonas aeruginosa (Table 1). Based on the primers, loop-mediated isothermal amplification technology was used to amplify the test samples to obtain amplification products. The amplification reaction system consisted of: 0.8 μL primer mixture (using nuclease-free ultrapure water as solvent, including the following components at final concentrations: 1.6 μM inner primer, 0.2 μM outer primer, and 0.4 μM loop primer), 1.6 μL 10 nM dNTPs, 3.2 μL 10 nM betaine, 1.6 μL template, 2.0 μL 10×Bst buffer, 0.8 μL 8 U / μL Bst polymerase, and 10 μL double-distilled water.

[0032] Amplification reaction procedure: Incubate at 65℃ for 20 min; (2) Take 10 μL of amplification product, 1.0 μL of 4 U / μL thermosensitive double-stranded specific enzyme, 2.0 μL of 10×dsDNasebuffer, and 7 μL of ultrapure water, mix them gently, place them in a qPCR instrument, incubate at 37℃ for 10 min to digest the amplification product, and perform fluorescence detection.

[0033] In this detection method, internal primer molecular probes are amplified via LAMP to form a double-stranded DNA product. Subsequently, when ezDNase is added to the reaction mixture, it immediately recognizes the dsDNA substrate and cleaves the amplified product upon activation. Figure 2 (a) The amplification products and digestion products described above were subjected to Native-PAGE (…). Figure 2 b) Detection, ultraviolet light irradiation ( Figure 2 c) and fluorescence detection ( Figure 2 (d) The results showed that the LAMP amplification products exhibited typical ladder-like bands, while the negative control region showed no amplification. After digestion with ezDNase, the molecular weight of the amplification products decreased, migrated below the primer bands, and aggregated together; this indicated that ezDNase successfully recognized and digested the amplification products. After irradiating the reaction solution with UV light, the positive reaction tube turned milky white, while the negative reaction tube remained clear. Simultaneously, under fluorescence detection, the positive reaction showed strong fluorescence, while the negative control showed no fluorescence signal; this indicates that the internal primer molecular probes, after amplification to form dsDNA, were successfully cleaved by ezDNase.

[0034] To more intuitively observe the cleavage process of the internal primer molecular probe, 10 μL of amino silica microspheres were placed in a 1.5 mL centrifuge tube and centrifuged at 8000 rpm for 10 min, discarding the supernatant. Then, 500 μL of enzyme-free water was added, and the mixture was vortexed and washed, followed by centrifugation to remove the supernatant. This process was repeated three times. 25 μL of pH 7.0 Tris-HCl (containing 0.1 mM DTT) was added to the reaction tube, and the microspheres were resuspended by vortexing. Then, 25 μL of the digestion product (negatively charged nucleic acids) was added, and the mixture was incubated at 37°C for 1 h in a constant-temperature shaking metal bath. After the reaction was complete, the mixture was centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the washing was repeated twice. Finally, 50 μL of enzyme-free water was added to resuspend the microspheres, and the morphology and surface fluorescence of the microspheres were observed under a confocal fluorescence microscope. Figure 2 (e) The results showed that strong fluorescence was observed on the surface of the microspheres that adsorbed the positively charged reaction products; while the single-stranded internal primer probes generated by the negative reaction were not degraded by ezDNase; the fluorescence remained in a quenched state, and no fluorescence was observed on the surface of the microspheres.

[0035] The above results demonstrate that the multiplex nucleic acid detection method established by combining LAMP technology with ezDNase is feasible.

[0036] Example 2 Performance Determination of Multiplex Nucleic Acid Detection Method

[0037] To determine the optimal reaction conditions for the multiplex nucleic acid detection method ezDNase, the incubation temperature and enzyme concentration were optimized. The incubation temperature was set to five different levels: 25, 25, 30, 35, 37, and 40°C. The results were measured at each level, and the signal-to-noise ratio (SNR) was calculated. SNR = Fluorescence value of positive experimental group / Fluorescence value of negative control group; where the positive experimental group is the group with added ezDNase and the negative control group is the group without added ezDNase. The results showed that the SNR was highest at 35℃, therefore 35℃ is the optimal reaction temperature for ezDNase. The enzyme concentration was set to five different levels: 1, 2, 3, 4, and 5 U, and tests were performed at each level. Therefore, 35℃ is the optimal incubation temperature and 4U is the optimal enzyme concentration.

[0038] Under optimal experimental conditions, the sensitivity and specificity of multiplex nucleic acid detection methods were determined.

[0039] Sensitivity and specificity determination: The LAMP and ezDNase-based multiplex nucleic acid detection method can perform enzymatic digestion without guide probes or other reagents, and the detection time is only 25 minutes (20 minutes for LAMP reaction and 5 minutes for ezDNase reaction). Figure 3(a and b in the text) Pseudomonas aeruginosa ( P. aeruginosa )of oprL Using genes as targets, nucleic acids were extracted from cultured standard strains as templates, and the concentration was set at 1 copy / μL~1.0×10⁻⁶. 6 Seven concentration gradients within the range of copies / μL were detected (the probes used for detection are shown in Table 1). The results are as follows: Figure 3 As shown in c; then specific detection was performed, and the results are as follows. Figure 4 As shown; Sensitivity results showed that the multiplex nucleic acid detection method had considerable detection sensitivity, all reaching 10 copies / μL. Specificity results showed that no pathogens such as Vibrio parahaemolyticus, Vibrio vulnificus, Klebsiella pneumoniae, methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli, Staphylococcus flexneri, Staphylococcus epidermidis, Staphylococcus aureus, Enterobacter faecalis, Staphylococcus agalactiae, or Staphylococcus tachyzoae were detected. Therefore, the multiplex nucleic acid detection method of this invention has excellent analytical sensitivity, meeting the requirements for nucleic acid diagnosis of infectious pathogens, and also exhibits good specificity when detecting Pseudomonas aeruginosa samples.

[0040] Then, referring to the above method, multiplex nucleic acid detection was used to detect Vibrio parahaemolyticus (V. parahaemolyticus). V. parahaemolyticus The sensitivity and specificity of the detection were assessed (primer sequences are shown in Table 1, and the results are as follows). Figure 5 (as shown) The results showed that the limit of detection (LOD) for Vibrio parahaemolyticus by the multiplex nucleic acid detection method was 10 copies / μL. In the specificity detection, both the positive control (using Vibrio parahaemolyticus nucleic acid as a template) and the negative control (using distilled water as a template) were correctly identified, and no other bacterial nucleic acids were detected. This indicates that the multiplex nucleic acid detection method has good sensitivity and specificity for detecting Vibrio parahaemolyticus.

[0041] Finally, the performance of multiplex nucleic acid detection method for detecting EBV virus was evaluated; the EBV nucleic acid template used in the experiment was a synthetic genome plasmid (primer sequences are shown in Table 1, and the results are as follows). Figure 6 (as shown) The results showed that the detection sensitivity of EBV reached 1 copy / μL. In the specific detection, herpes simplex virus type 1 (HSV-1), varicella-zoster virus (VZV), cytomegalovirus (CMV), and several other viruses were not detected. The positive and negative control tests were correct, indicating that the multiplex nucleic acid detection method has good sensitivity and specificity for detecting EBV.

[0042] The above experimental results show that the multiplex nucleic acid detection method of the present invention exhibits good sensitivity and specificity when applied to a variety of different pathogens, and has strong versatility.

[0043] Multiple detection: ezDNase also exhibits a non-sequence-specific cleavage mechanism. Due to its preference for double-stranded DNA, it can precisely cleave reporter probes labeled with different fluorescent tags, thus enabling the development of multiplex nucleic acid methods with multiplex detection capabilities. Figure 3 (as shown in d in the table). To verify the multiplex detection capability, three different human papillomavirus subtypes (HPV16, HPV18, and HPV52) were selected for detection. For the L1 gene plasmids of different subtypes, three sets of corresponding LAMP primers were designed, and the internal primer probes were modified with three different fluorophores (FAM, VIC, and Cy5) (as shown in Table 1) to distinguish different targets. Furthermore, these primers were mixed in the same reaction tube, and various combinations of single, double, and triple target configurations were designed for verification. The fluorescence detection results are shown in Table 1. Figure 3 The 'e' in the figure and Figure 7 As shown; Fluorescence detection results showed that all target combinations produced corresponding fluorescence amplification curves in the same reaction solution, and all targets were correctly detected.

[0044] Subsequently, the enzyme digestion products were adsorbed onto the surface of amino silica microspheres, and the fluorescence was directly observed under a confocal fluorescence microscope. Figure 3 f); The results showed that the digestion products of the three different HPV subtypes exhibited fluorescent spots of different colors; for the digestion products with two and three targets, two and three corresponding fluorescent spots were observed, respectively. The above results demonstrate that multiplex nucleic acid detection methods can detect multiple targets simultaneously.

[0045] Table 1 Primer sequences for detecting pathogen nucleic acids based on multiplex nucleic acid detection methods

[0046] Example 3 Clinical Sample Testing

[0047] To verify the diagnostic capability of multiplex nucleic acid detection methods for pathogens, this study collected and tested a large number of clinical samples.

[0048] The multiplex nucleic acid detection method of this invention was used to detect 79 clinical samples of Pseudomonas aeruginosa, 21 clinical samples of Vibrio parahaemolyticus, and 51 clinical samples of EBV. The detection results are as follows: Figure 8 As shown; Because nucleic acid extraction is complex and time-consuming, it will significantly prolong the overall analysis time, which is not suitable for POCT testing environments for infectious diseases. Therefore, in the sample processing, nucleic acid extraction is not performed. Instead, nucleic acid lysis buffer is used, and the lysis process can be completed in just 1 to 3 minutes. The results showed that the sensitivity and negative predictive value of Pseudomonas aeruginosa were 97.9% and 96.9%, respectively, while the specificity and positive predictive value were both 100%. Vibrio parahaemolyticus and EBV were correctly distinguished in all samples, with both sensitivity and specificity at 100%. Therefore, the multiplex nucleic acid detection method can sensitively and specifically detect a variety of complex samples and pathogens, making it a rapid, accurate, and universal diagnostic tool for infectious diseases.

[0049] The diagnostic performance of multiplex nucleic acid detection methods in multiple applications was evaluated using cervical samples. 183 cervical samples were collected, tested using multiplex nucleic acid detection methods, and the results were further analyzed. The results are as follows: Figure 9 As shown; The results showed that the HPV16, HPV18, and HPV52 detection primers could correctly identify the corresponding HPV samples and had good specificity for samples of other HPV subtypes. The detection sensitivity of the three HPV subtypes was greater than 93.9%, the negative predictive value was greater than 94.2%, and the specificity and positive predictive value were both 100%. Therefore, the multiplex nucleic acid detection method has been successfully applied to HPV genotyping, and its multiplex detection capability has great potential in practical applications, especially in pathogen differentiation and subtype detection.

[0050] As can be seen from the above embodiments, the present invention provides a multiplex nucleic acid detection method based on loop-mediated isothermal amplification (LAMP) technology and nuclease. This method combines LAMP and ezDNase, which not only shortens the detection time and reduces the detection cost, but also has high sensitivity and specificity, enabling multiplex detection.

[0051] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A multiplex nucleic acid detection method based on loop-mediated isothermal amplification technology and nuclease, characterized in that, Includes the following steps: (1) Primers were designed for the target, and loop-mediated isothermal amplification technology was used to amplify the test sample based on the primers to obtain the amplification product; (2) The amplified product was digested with a thermosensitive double-stranded specific enzyme and then detected by fluorescence.

2. The multiplex nucleic acid detection method according to claim 1, characterized in that, In step (1), the primers include inner primers, outer primers, and loop primers; The inner primers are modified with fluorescent groups and quenching groups.

3. The multiplex nucleic acid detection method according to claim 2, characterized in that, The fluorescent groups include FAM, VIC, and CY5; the quenching groups include BHQ1 and BHQ2.

4. The multiplex nucleic acid detection method according to claim 1, characterized in that, In step (1), the amplification reaction system consists of 0.6-1.0 μL primer mixture, 1.4-1.8 μL dNTPs, 3.0-3.4 μL betaine, 1.4-1.8 μL template, 1.8-2.2 μL 10×Bst buffer, 0.6-1.0 μL Bst polymerase, and 8.8-11.2 μL water.

5. The multiplex nucleic acid detection method according to claim 4, characterized in that, The primer mixture, using nuclease-free ultrapure water as a solvent, comprises the following components at final concentrations: 1.4–1.8 μM inner primer, 0.1–0.3 μM outer primer, and 0.2–0.6 μM loop primer; The concentration of the dNTPs is 8-12 nM; the concentration of the betaine is 8-12 nM; and the concentration of the Bst polymerase is 6-10 U / μL.

6. The multiplex nucleic acid detection method according to claim 1, characterized in that, In step (1), the amplification reaction procedure is: incubation at 60~70℃ for 15~25 min.

7. The multiplex nucleic acid detection method according to claim 1, characterized in that, In step (2), the digestion reaction system is: 8~12μL amplification product, 0.8~1.2μL thermosensitive double-stranded specific enzyme, 1.8~2.2μL 10×dsDNase buffer, and 4.6~9.4μL water.

8. The multiplex nucleic acid detection method according to claim 7, characterized in that, The concentration of the thermosensitive double-stranded specific enzyme is 1~5 U / μL.

9. The multiplex nucleic acid detection method according to claim 1, characterized in that, In step (2), the digestion reaction procedure is: incubation at 25~40℃ for 5~20 min.

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