WO2022193754A1 - 荧光可视化的lamp产物检测方法及其试剂盒和应用 - Google Patents

荧光可视化的lamp产物检测方法及其试剂盒和应用 Download PDF

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WO2022193754A1
WO2022193754A1 PCT/CN2021/138124 CN2021138124W WO2022193754A1 WO 2022193754 A1 WO2022193754 A1 WO 2022193754A1 CN 2021138124 W CN2021138124 W CN 2021138124W WO 2022193754 A1 WO2022193754 A1 WO 2022193754A1
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detection
isothermal amplification
aggregation
primers
reaction
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French (fr)
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张鹏飞
王媛媛
蔡林涛
龚萍
李菁
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深圳先进技术研究院
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Definitions

  • the invention relates to the field of pathogenic microorganism detection, in particular to a fluorescent visualization LAMP (loop-mediated isothermal amplification) product detection method, a kit and application thereof.
  • LAMP loop-mediated isothermal amplification
  • LAMP loop mediated isothermal amplification
  • the reaction can produce a large number of amplification products, namely magnesium pyrophosphate white precipitate. Therefore, the experimenter can judge the existence of the target gene by observing the degree of the white turbid precipitate with the naked eye.
  • a stable heat source such as a water bath or a metal bath
  • It has the characteristics of strong specificity; the detection cost is much lower than that of fluorescent quantitative PCR, and it has a very broad application prospect.
  • the detection of bacteria and viruses mainly includes traditional detection and molecular biological detection.
  • Traditional methods are time-consuming and labor-intensive, and the reliability of detection results is low.
  • the sensitivity of immunological detection technology is low, and the production process of antibodies is time-consuming and complicated.
  • the detection cost is high.
  • PCR technology is fast and accurate, it requires expensive equipment and the detection cost is high, so it is not suitable for promotion and application in grassroots departments. Therefore, there is an urgent need for a simple, rapid, high-sensitivity, and specific method for detecting pathogenic microorganisms that does not require expensive instruments.
  • the present invention proposes a fluorescent visualization LAMP (loop-mediated isothermal amplification) product detection method and kit.
  • the detection method of the present invention adds aggregation to the loop-mediated isothermal amplification system.
  • the luminescent dye is induced, and after the reaction is completed, the reaction result is judged by observing with an ultraviolet lamp.
  • the kit of the invention adds aggregation-induced luminescent dyes on the basis of the LAMP kit, and can rapidly and accurately detect pathogenic microorganisms and transgenic animals and plants.
  • the invention provides a fluorescent visualization method for detecting a loop-mediated isothermal amplification product.
  • Aggregation-induced luminescent dye is added to the loop-mediated isothermal amplification system, and after the reaction is completed, the reaction result is judged by observing with an ultraviolet lamp.
  • the invention combines the aggregation-induced luminescent dye with the traditional LAMP technology, which can improve the sensitivity and specificity, and realize the fluorescence visualization detection.
  • the temperature of the reaction is 60-65° C.
  • the time of the reaction is 15-40 min.
  • the aggregation-induced luminescent dye is added before the loop-mediated isothermal amplification step.
  • the cap is opened and added for the reaction, which can overcome the problem of aerosol contamination and can realize detection without opening the cap.
  • the method for judging the reaction result is as follows: if fluorescence occurs under the ultraviolet lamp, it is positive; if there is no fluorescent signal under the ultraviolet lamp, it is negative. If the corresponding fragment is amplified in the reaction system, the aggregation-induced luminescent dye can combine with the substance in the reaction system to emit fluorescence, thereby showing fluorescence under an ultraviolet lamp.
  • the invention also provides a fluorescence visualization loop-mediated isothermal amplification product detection kit, comprising the following components: dNTP, MgSO 4 , isothermal amplification buffer, Bst DNA polymerase, aggregation-induced luminescent dye, and double distilled water.
  • the present invention also provides a novel coronavirus detection kit, comprising the following components: dNTP, MgSO 4 , isothermal amplification buffer, Bst DNA polymerase, aggregation-induced luminescent dye, double distilled water, and primers;
  • the primers include 4, and their sequences are GCCAAAAGGCTTCTACGCA, TTTGGCCTTGTTGTTGTTGG, TCCCCTACTGCTGCCTGGAGGCAGTCAAGCCTCTTCTCG, TCCTGCTAGAATGGCTGGCAATTTTGCTCTCAAGCTGGTTCA, respectively, in the 5'-3' direction.
  • the above sequence takes the nucleic acid sequence with high conservation and strong specificity in the nucleocapsid gene of the novel coronavirus as the target sequence.
  • the invention also provides an Escherichia coli detection kit, comprising the following components: dNTP, MgSO 4 , isothermal amplification buffer, Bst DNA polymerase, aggregation-induced luminescent dye, double distilled water, and primers; the primers include 6,
  • the 5'-3' directions of the sequences are CACCTTCATGGATATCGAGATT, TGGAGGATTTAAGCCATCTC, CGAGCGTACAGCTGCAAAATGATATCTTTCGATACCACGACCT, CCCTTCTCCCTTTGTAACAAGATGACGCATAGTCAGCCCAT, TACGAAAGCCTGGGGCG, CCTGTCATCGACAGCAACATTCA.
  • the above primers use nucleic acid sequences with high conservation and high specificity in Escherichia coli as target sequences. The nucleic acid sequence was obtained after screening a large number of documents.
  • the present invention also provides a Staphylococcus aureus detection kit, comprising the following components: dNTP, MgSO 4 , isothermal amplification buffer, Bst DNA polymerase, aggregation-induced luminescent dye, double distilled water, and primers; the primers include 6
  • the 5'-3' directions of the sequences are GACGATTGTTTGCTTGTTCT, CCAAGAGATAGCCCCTAGA, GAGTGTGAATAAATATGGCTGTTCGTAGCAGCAATGCATCCAA, AATGGTATTAGTCGCATTACCCGAGCAGAAGAAATTCTAAAACTCA, AAAGTTACTGTGTTTGGACCTG, CACTTATTTTAGCTATATTCAATAA.
  • the present invention also provides a Shigella detection kit, comprising the following components: dNTP, MgSO 4 , isothermal amplification buffer, Bst DNA polymerase, aggregation-induced luminescent dye, double distilled water, and primers; the primers include 6 , the 5'-3' directions of the sequences are ATACCGTCTCTGCACGCA, GCCTTCTGATGCCTGATGG, CTGTCGAAGCTCCGCAGAGGGGATTCCGTGAACAGGTCG, AGTCTTTCGCTGCTGCTGCTGATCCGGAGATTGTTCCATGTG, GAGTTTTTCCAGCCATGCAG, TGCCACTGAGAGCTGTGAG, respectively.
  • the present invention also provides a human papillomavirus detection kit, comprising the following components: dNTP, MgSO 4 , isothermal amplification buffer, Bst DNA polymerase, aggregation-induced luminescent dye, double distilled water, and primers; the primers include 6
  • the 5'-3' directions of the sequences are GCACCAAAAGAGAACTGC, AGCATATGGATTCCCATCTC, TTGTTTGCAGCTGGGGAATTTATTTCAGGACCCAACAGGAG, TTAGAATGTGTGTACTGCAAGCATCCCGAAAAGCAAAGTCA, GGTAACTTTCTGGGTCGCTCC, AGTTACTGCGACGTGAGGT, respectively.
  • the invention also provides the application of the detection method in the detection of pathogenic microorganisms and the detection of transgenes.
  • the pathogenic microorganisms include viruses and bacteria.
  • viruses and bacteria such as the new coronavirus, Escherichia coli, Staphylococcus aureus, Shigella and human papillomavirus.
  • the present invention achieves the following technical effects:
  • the detection method of the present invention adds aggregation-induced luminescent dye on the basis of LAMP technology, which improves the detection sensitivity, and the detection limit is 10 copies/tube.
  • the detection method of the present invention can shorten the detection time, and the target gene can be clearly detected in at least 15 minutes.
  • the detection method of the present invention does not require subsequent electrophoresis detection. Under the ultraviolet lamp, the reaction system emits fluorescence as positive, and the color does not emit light as negative. The fluorescence visualization is realized, and the result judgment is intuitive, accurate, and simple.
  • the aggregation-induced luminescent dye is added to the LAMP reaction system in advance, which overcomes the problem of aerosol pollution and realizes detection without opening the lid.
  • the detection kit of the present invention can quickly and accurately detect pathogenic microorganisms and transgenic animals and plants, and has broad application prospects in disease detection and animal husbandry.
  • Figure 1 is the verification of the effect of the primer combination of Example 1 for amplifying the novel coronavirus.
  • Figure 2 shows the verification of the results of rapid detection of new coronavirus using AIE dye in Example 2.
  • FIG. 3 is the sensitivity detection result of the detection method of the present invention in Example 3.
  • FIG. 3 is the sensitivity detection result of the detection method of the present invention in Example 3.
  • FIG. 4 is the verification of the amplification effect of the primer combination of Example 4 on Escherichia coli.
  • FIG. 5 is the verification of the results of rapid detection of Escherichia coli using AIE dye in Example 5.
  • FIG. 5 is the verification of the results of rapid detection of Escherichia coli using AIE dye in Example 5.
  • FIG. 6 is a verification of the effect of the primer combination of Example 6 for amplifying Staphylococcus aureus.
  • FIG. 7 is a verification of the effect of the primer combination of Example 7 for amplifying Shigella.
  • FIG. 8 is the verification of the effect of the primer combination of Example 8 in amplifying human papillomavirus.
  • AIE Aggregation-Induced Emission
  • the invention utilizes the characteristic that AIE dye can emit fluorescence when combined with substances in the LAMP reaction system, and designs a fluorescent visualization
  • LAMP loop-mediated isothermal amplification
  • the following examples illustrate the operation steps and result determination method of the detection method of the present invention by taking the detection of new coronavirus, Escherichia coli, Staphylococcus aureus, Shigella and human papillomavirus as examples, because the method of the present invention is based on the detection of nucleic acid fragments As long as specific primers are designed for certain viruses and bacteria, it is not limited to the above pathogenic microorganisms, other pathogenic microorganisms and transgenic animals and plants are applicable.
  • TTAPE 1,1,2,2-tetra[4-(2-triethylaminoethoxy)phenyl]tetrabromoethylene
  • AIE dyes have similar properties, all are positively charged, and all can bind to negatively charged DNA, so those skilled in the art can infer that other AIE dyes can be applied to the following scheme.
  • the nucleic acid sequences with high conservation and strong specificity in the nucleocapsid gene of the novel coronavirus were screened out by referring to the literature as target sequences, and the online software Primer Explorer v5 was used to design LAMP primers were synthesized and the following primers were obtained (5'-3' direction):
  • Outer primer F3 GCCAAAAGGCTTCTACGCA;
  • Outer primer B3 TTTGGCCTTGTTGTTGTTGG;
  • Inner primer FIP TCCCCTACTGCTGCCTGGAGGCAGTCAAGCCTCTTCTCG;
  • Inner primer BIP TCCTGCTAGAATGGCTGGCAATTTTGCTCTCAAGCTGGTTCA.
  • the nucleic acid of the new coronavirus was added to tube 1 as a template, and the tube 2 was not added, and water was used as a negative control.
  • the reaction system was as follows (total volume was 25 ⁇ L), the aggregation-induced luminescent dye was added prior to the loop-mediated isothermal amplification step.
  • the reaction temperature is 60-65°C, heated in a water bath, and the reaction time is 20-40min.
  • Example 2 AIE dye rapid amplification test
  • Outer primer F3 GCCAAAAGGCTTCTACGCA;
  • Outer primer B3 TTTGGCCTTGTTGTTGTTGG;
  • Inner primer FIP TCCCCTACTGCTGCCTGGAGGCAGTCAAGCCTCTTCTCG;
  • Inner primer BIP TCCTGCTAGAATGGCTGGCAATTTTGCTCTCAAGCTGGTTCA.
  • the nucleic acid of the new coronavirus was added to the tubes 1-10 as a template, and the tube 11 was not added, and replaced with water as a negative control.
  • the reaction system is as follows (total volume is 25 ⁇ L):
  • Amplify in a water bath set ten time gradients of 1min, 2min, 3min, 4min, 5min, 10min, 15min, 20min, 30min, and 40min, and the temperature is 64°C.
  • Tube 7 was amplified at 64°C for 10min, tube 7 was amplified at 64°C for 15min, tube 8 was amplified at 64°C for 20min, tube 9 was amplified at 64°C for 30min, tube 10 was amplified at 64°C for 40min, and tube 11 was a negative control.
  • the results show that blue fluorescence starts to appear from tube 7, and the fluorescence intensity from tube 7 to tube 10 is getting stronger and stronger, indicating that the detection method of the present invention can obviously detect the target gene of the virus in 15 minutes under the condition of 64 ° C, which can greatly shorten the detection time. time.
  • Embodiment 3 Sensitivity test of detection method of the present invention
  • novel coronavirus-specific gene sequence nucleocapsid(N) was diluted 10-fold, and the reaction system and conditions of the visualized LAMP detection method were carried out as follows.
  • Outer primer F3 GCCAAAAGGCTTCTACGCA;
  • Outer primer B3 TTTGGCCTTGTTGTTGTTGG;
  • Inner primer FIP TCCCCTACTGCTGCCTGGAGGCAGTCAAGCCTCTTCTCG;
  • Inner primer BIP TCCTGCTAGAATGGCTGGCAATTTTGCTCTCAAGCTGGTTCA.
  • the nucleic acid of the new coronavirus was added to the tubes 1-8 as a template, and the tube 9 was not added and replaced with water as a negative control.
  • the reaction system is as follows (total volume is 25 ⁇ L):
  • the reaction temperature is 60-65°C, heated in a water bath, and the reaction time is 20-40min.
  • Outer primer F3 CACCTTCATGGATATCGAGATT;
  • Outer primer B3 TGGAGGATTTAAGCCATCTC;
  • Inner primer FIP CGAGCGTACAGCTGCAAAATGATATCTTTCGATACCACGACCT;
  • Inner primer BIP CCCTTCTCCCTTTGTAACAAGATGACGCATAGTCAGCCCAT;
  • Loop primer LF TACGAAAGCCTGGGGCG
  • Loop primer LB CCTGTCATCGACAGCAACATTCA.
  • the nucleic acid of Escherichia coli was added to tube 1 as a template, and the tube 2 was not added, and water was used instead, as a negative control.
  • the reaction system is as follows (total volume is 25 ⁇ L):
  • the reaction temperature is 60-65°C, heated in a water bath, and the reaction time is 20-40min.
  • Example 5 AIE dye rapid amplification Escherichia coli test
  • Outer primer F3 CACCTTCATGGATATCGAGATT;
  • Outer primer B3 TGGAGGATTTAAGCCATCTC;
  • Inner primer FIP CGAGCGTACAGCTGCAAAATGATATCTTTCGATACCACGACCT;
  • Inner primer BIP CCCTTCTCCCTTTGTAACAAGATGACGCATAGTCAGCCCAT;
  • Loop primer LF TACGAAAGCCTGGGGCG
  • Loop primer LB CCTGTCATCGACAGCAACATTCA.
  • the nucleic acid of Escherichia coli was added to tubes 2-11 as a template, and the tube 1 was not added, but water was used as a negative control.
  • the reaction system is as follows (total volume is 25 ⁇ L):
  • Amplify in a water bath set ten time gradients of 1min, 2min, 3min, 4min, 5min, 10min, 15min, 20min, 30min, and 40min, and the temperature is 64°C.
  • Tube 2 was amplified at 64°C for 1 min
  • tube 3 was amplified at 64°C for 2 min
  • tube 4 was amplified at 64°C for 3 min
  • tube 5 was amplified at 64°C for 4 min
  • tube 6 was amplified at 64°C for 5 min
  • tube 7 was amplified at 64°C Amplification at °C for 10 min
  • tube 8 for 15 min at 64 °C
  • tube 9 for 20 min at 64 °C
  • tube 10 for 30 min at 64 °C
  • tube 11 for 40 min at 64 °C.
  • Embodiment 6 Staphylococcus aureus gene amplification detection
  • Outer primer F3 GACGATTGTTTGCTTGTTCT;
  • Outer primer B3 CCAAGAGATAGCCCCTAGA;
  • Inner primer FIP GAGTGTGAATAAATATGGCTGTTCGTAGCAGCAATGCATCCAA;
  • Inner primer BIP AATGGTATTAGTCGCATTACCCGAGCAGAAGAAATTCTAAAACTCA; loop primer LF: AAAGTTACTGTGTTTGGACCTG;
  • Loop primer LB CACTTATTTTAGCTATATTCAATAA.
  • the nucleic acid of Staphylococcus aureus was added to tube 1 as a template, and the tube 2 was not added, and water was used instead, as a negative control.
  • the reaction system is as follows (total volume is 25 ⁇ L):
  • the reaction temperature is 60-65°C, heated in a water bath, and the reaction time is 20-40min.
  • Outer primer F3 ATACCGTCTCTGCACGCA;
  • Outer primer B3 GCCTTCTGATGCCTGATGG;
  • Inner primer FIP CTGTCGAAGCTCCGCAGAGGGGATTCCGTGAACAGGTCG;
  • Inner primer BIP AGTCTTTCGCTGCTGCTGCTGATCCGGAGATTGTTCCATGTG;
  • Loop primer LF GAGTTTTCAGCAGCAGCAG
  • Loop primer LB TGCCACTGAGAGCTGTGAG.
  • the reaction system is as follows (total volume is 25 ⁇ L):
  • dNTPs 10mM 3.5 1.4mM MgSO 4 100mM 1.5 6mM Isothermal Amplification Buffer 10X 2.5 1X Bst DNA polymerase 8U/ ⁇ L 1 0.32U/ ⁇ L aggregation-induced luminescent dyes 1 double distilled water Make up to 25 ⁇ L
  • the reaction temperature is 60-65°C, heated in a water bath, and the reaction time is 20-40min.
  • Example 8 Human papillomavirus gene amplification detection
  • Outer primer F3 GCACCAAAAGAGAACTGC
  • Outer primer B3 AGCATATGGATTCCCATCTC;
  • Inner primer FIP TTGTTTGCAGCTGGGGAATTTATTTCAGGACCCAACAGGGAG;
  • Inner primer BIP TTAGAATGTGTGTACTGCAAGCATCCCGAAAAGCAAAGTCA;
  • Loop primer LF GGTAACTTTCTGGGTCGCTCC
  • Loop primer LB AGTTACTGCGACGTGAGGT.
  • the human papillomavirus nucleic acid was added to tube 1 as a template, and the tube 2 was not added, and water was used instead, as a negative control.
  • the reaction system is as follows (total volume is 25 ⁇ L):
  • the reaction temperature is 60-65°C, heated in a water bath, and the reaction time is 20-40min.
  • the detection method of the present invention can not only be used to detect pathogenic microorganisms such as bacteria and viruses, but also can be used to detect whether genes have been transferred into animals and plants.
  • the operation method is similar to the detection of bacteria and viruses, except that the primer fragments are replaced with primers targeting specific nucleic acid sequences in transgenic animals and plants, and the genomic DNA of the animals and plants to be tested is extracted, and the operation steps and detection methods of the foregoing embodiments are utilized, If there is fluorescence observed under the ultraviolet light, it means that the gene has been transferred into the animal or plant to be tested, and it is a transgenic animal or plant. If there is no fluorescent signal, it is a non-transgenic animal or plant.
  • the present invention discloses a fluorescent visualization loop-mediated isothermal amplification (LAMP) product detection method, kit and application thereof.
  • the detection method of the present invention adds aggregation-induced luminescence to the loop-mediated isothermal amplification system.
  • AIE dye
  • the kit of the invention adds aggregation-induced luminescent dye on the basis of the LAMP kit, which improves the detection sensitivity, the detection limit is 10 copies/tube, and the detection time can be shortened, and the detection can be obviously detected in at least 15 minutes. target gene.
  • the detection method of the present invention does not require subsequent electrophoresis detection, and the reaction system emits blue fluorescence under the ultraviolet lamp as positive, and the color does not emit light as negative, realizing fluorescence visualization, and the result judgment is intuitive, accurate, and simple.
  • the aggregation-induced luminescent dye is added to the LAMP reaction system in advance, which overcomes the problem of aerosol pollution, realizes the detection without opening the lid, and can detect pathogenic microorganisms and transgenic animals and plants quickly and accurately.

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Abstract

本发明提供一种荧光可视化的环介导等温扩增(LAMP)产物检测方法及其试剂盒和应用，包括在环介导等温扩增体系中添加了聚集诱导发光染料(AIE)，反应结束后，通过紫外灯观察，判断反应结果。

Description

荧光可视化的LAMP产物检测方法及其试剂盒和应用 技术领域
本发明涉及病原微生物检测领域，特别涉及一种荧光可视化的LAMP(环介导等温扩增)产物检测方法及其试剂盒和应用。
背景技术
随着现代分子技术的发展，开发了许多核酸扩增技术。其中，日本学者Notomi于2000年在Nucleic Acids Res杂志上公开了环介导等温扩增技术(Loop mediated isothermal amplification，LAMP)，并已成功应用于SARS、禽流感、HIV等疾病的检测中。LAMP是针对靶序列上6或8个特异区域设计4或6条特异引物，在链置换型DNA聚合酶(Bst DNA polymerase)的作用下，60-65℃恒温扩增，60分钟左右即可实现指数级别的核酸扩增，是一种“简便、快速、精确、低价”的基因扩增方法。反应能产生大量的扩增产物即焦磷酸镁白色沉淀，因此，实验人员可以通过肉眼观察白色浑浊沉淀的程度，来判断靶基因是否存在。由于LAMP反应是在恒温条件下进行的，所以一个稳定的热源(例如水浴或者金属浴)就能满足实验条件的要求，不需要模板的热变性、温度循环、电泳等过程，具有简单、快速、特异性强的特点；检测成本远低于荧光定量PCR，具有极为广泛的应用前景。
目前，细菌和病毒的检测主要有传统检测和分子生物学检测法等，传统方法耗时耗力，且检测结果可靠性低，免疫学检测技术的灵敏度低，且抗体的制作过程耗时、复杂，检测成本高，PCR技术虽然快速、准确，但需要昂贵的仪器设备，检测成本高，不适宜在基层部门推广应用。所以目前亟需一种简单快速、灵敏度高、特异性强、不需要借助昂贵仪器的检测病原微生物的方法。
发明内容
针对现有技术中的缺陷，本发明提出了一种荧光可视化的LAMP(环介导等温扩增)产物检测方法及试剂盒，本发明的检测方法在环介导等温扩增体系中添加了聚集诱导发光染料，反应结束后，通过紫外灯观察，判断反应结果。本发明的试剂盒在LAMP试剂盒的基础上添加聚集诱导发光染料，可快速、精确的检测病原微生物和转基因动植物。
本发明提供一种荧光可视化的环介导等温扩增产物的检测方法，在环介导等温扩增体系中添加聚集诱导发光染料，反应结束后，通过紫外灯观察，判断反应结果。本发明将聚集诱导发光染料与传统的LAMP技术结合，能够提高灵敏度和特异性，实现荧光可视化检测。
进一步的，所述反应的温度为60-65℃，所述反应的时间为15-40min。
进一步的，所述的聚集诱导发光染料在环介导等温扩增步骤之前加入。区别于环介导等温扩增步骤结束后再开盖加入进行反应，可以克服气溶胶污染的问题，能够实现不开盖检测。
进一步的，所述反应结果的判断方法如下：紫外灯下出现荧光，即为阳性；紫外灯下没有荧光信号，即为阴性。如果反应体系中扩增出相应片段，聚集诱导发光染料则能够与反应体系中的物质结合发出荧光，从而在紫外灯下显出荧光。
本发明还提供一种荧光可视化的环介导等温扩增产物检测试剂盒，包括如下成份：dNTP、MgSO 4、等温扩增缓冲液、Bst DNA聚合酶、聚集诱导发光染料、双蒸水。
本发明还提供一种新冠病毒检测试剂盒，包括如下成份：dNTP、MgSO 4、等温扩增缓冲液、Bst DNA聚合酶、聚集诱导发光染料、双蒸水、引物；
所述引物包含4个，其序列5’-3’方向分别为GCCAAAAGGCTTCTACGCA、TTTGGCCTTGTTGTTGTTGG、TCCCCTACTGCTGCCTGGAGGCAGTCAAGCCTCTTCTCG、 TCCTGCTAGAATGGCTGGCAATTTTGCTCTCAAGCTGGTTCA。以上序列是以新型冠状病毒的nucleocapsid基因中保守性高、特异性强的核酸序列作为靶序列。
本发明还提供一种大肠杆菌检测试剂盒，包括如下成份：dNTP、MgSO 4、等温扩增缓冲液、Bst DNA聚合酶、聚集诱导发光染料、双蒸水、引物；所述引物包含6个，其序列5’-3’方向分别为CACCTTCATGGATATCGAGATT、TGGAGGATTTAAGCCATCTC、CGAGCGTACAGCTGCAAAATGATATCTTTCGATACCACGACCT、CCCTTCTCCCTTTGTAACAAGATGACGCATAGTCAGCCCAT、TAACGAAAGCCTGGGGCG、CCTGTCATCGACAGCAACATTCA。上述的引物是以大肠杆菌中保守性高、特异性强的核酸序列作为靶序列。该核酸序列通过查阅大量文献筛选后得出。
本发明还提供一种金黄色葡萄球菌检测试剂盒，包括如下成份：dNTP、MgSO 4、等温扩增缓冲液、Bst DNA聚合酶、聚集诱导发光染料、双蒸水、引物；所述引物包含6个，其序列5’-3’方向分别为GACGATTGTTTGCTTGTTCT、CCAAGAGATAGCCCCTAGA、GAGTGTGAATAAATATGGCTGTTCGTAGCAGCAATGCATCCAA、AATGGTATTAGTCGCATTACCCGAGCAGAAGAAATTCTAAAACTCA、AAAGTTACTGTGTTTGGACCTG、CACTTATTTTAGCTATATTCAATAA。
本发明还提供一种志贺菌检测试剂盒，包括如下成份：dNTP、MgSO 4、等温扩增缓冲液、Bst DNA聚合酶、聚集诱导发光染料、双蒸水、引物；所述引物包含6个，其序列5’-3’方向分别为ATACCGTCTCTGCACGCA、GCCTTCTGATGCCTGATGG、CTGTCGAAGCTCCGCAGAGGGGATTCCGTGAACAGGTCG、AGTCTTTCGCTGCTGCTGCTGATCCGGAGATTGTTCCATGTG、GAGTTTTTCCAGCCATGCAG、TGCCACTGAGAGCTGTGAG。
本发明还提供一种人乳头瘤病毒检测试剂盒，包括如下成份：dNTP、 MgSO 4、等温扩增缓冲液、Bst DNA聚合酶、聚集诱导发光染料、双蒸水、引物；所述引物包含6个，其序列5’-3’方向分别为GCACCAAAAGAGAACTGC、AGCATATGGATTCCCATCTC、TTGTTTGCAGCTGGGGAATTTATTTCAGGACCCAACAGGAG、TTAGAATGTGTGTACTGCAAGCATCCCGAAAAGCAAAGTCA、GGTAACTTTCTGGGTCGCTCC、AGTTACTGCGACGTGAGGT。
本发明还提供所述的检测方法在病原微生物检测和转基因检测方面的应用。
进一步的，所述病原微生物包括病毒、细菌。如新冠病毒，大肠杆菌、金黄色葡萄球菌、志贺菌和人乳头瘤病毒。
综上，与现有技术相比，本发明达到了以下技术效果：
1.本发明的检测方法在LAMP技术的基础上添加了聚集诱导发光染料，提高了检测的灵敏度，检测限为10拷贝数/管。
2.本发明的检测方法能够缩短检测的时间，最少只需15分钟就能明显检测到目标基因。
3.本发明的检测方法无需后续的电泳检测，在紫外灯下反应体系发出荧光为阳性，颜色不发光为阴性，实现了荧光可视化，结果判定直观、准确，简单明了。
4.聚集诱导发光染料提前加入到LAMP反应体系中，克服了气溶胶污染的问题，实现了不开盖检测。
5.本发明的检测试剂盒能够快速、精确的检测病原微生物和转基因动植物，在疾病检测和畜牧业上具有广阔应用前景。
附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的某些 实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为实施例1的引物组合扩增新冠病毒效果验证。
图2为实施例2中使用AIE染料快速检测新冠病毒结果验证。
图3为实施例3中本发明的检测方法的灵敏度检测结果。
图4为实施例4的引物组合扩增大肠杆菌效果验证。
图5为实施例5中使用AIE染料快速检测大肠杆菌结果验证。
图6为实施例6的引物组合扩增金黄色葡萄球菌效果验证。
图7为实施例7的引物组合扩增志贺菌效果验证。
图8为实施例8的引物组合扩增人乳头瘤病毒效果验证。
具体实施方式
为了使本技术领域的人员更好地理解本发明方案，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分的实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例，都应当属于本发明保护的范围。
与常规染料相比，聚集诱导发光(Aggregation-Induced Emission，AIE)染料在分子溶解状态下显示弱荧光或无荧光。但是，当它们处于聚集状态以限制分子内自由旋转时，可以观察到强荧光。AIE材料的优势主要体现在：低背景、信噪比高、灵敏度好、抗光漂白能力强等方面。DNA带负电荷，AIE染料带正电荷，通过吸附作用可以使AIE染料与PCR产物结合，本发明利用了AIE染料与LAMP反应体系中的物质结合可以发出荧光的特性，设计出一种荧光可视化的环介导等温扩增(LAMP)产物检测方法及其试剂盒，用来快速、精确的检测病原微生物和转基因动植物，相比于LAMP 技术本身，本发明的方法提高了检测的灵敏度、缩短了检测时间，解决了目前病原微生物检测灵敏性不够高、特异性不够好、耗时长，需要借助复杂仪器的问题。
以下实施例以新冠病毒、大肠杆菌、金黄色葡萄球菌、志贺菌和人乳头瘤病毒的检测为例说明本发明的检测方法的操作步骤和结果判定方法，由于本发明的方法基于对核酸片段的检测，只要针对某种病毒和细菌设计出特异性的引物都可以使用，并不局限于上述病原微生物，其他病原微生物及转基因动植物均适用。
以下实施例中使用的聚集诱导发光染料为1,1,2,2-四[4-(2-三乙基氨乙氧基)苯基]四溴乙烯(TTAPE)，其结构式如下所示：
Figure PCTCN2021138124-appb-000001
但是其他AIE染料都具有相似的性质，均带正电荷，都能够与带负电荷的DNA结合，因此本领域技术人员能够推及其他AIE染料均可适用以下的方案。
实施例1新型冠状病毒基因扩增检测
为了验证本发明的检测方法可以运用于病毒的检测且有效，通过查阅文献筛选出新型冠状病毒的nucleocapsid基因中保守性高、特异性强的核酸序列作为靶序列，使用在线软件Primer Explorer v5设计出LAMP引物并合成，得到如下引物(5’-3’方向)：
外引物F3：GCCAAAAGGCTTCTACGCA；
外引物B3：TTTGGCCTTGTTGTTGTTGG；
内引物FIP：TCCCCTACTGCTGCCTGGAGGCAGTCAAGCCTCTTCTCG；
内引物BIP：TCCTGCTAGAATGGCTGGCAATTTTGCTCTCAAGCTGGTTCA。
分为2组，管1中加入新型冠状病毒的核酸作为模板，管2中不加入，以水代替，作为阴性对照。
反应体系如下(总体积为25μL)，聚集诱导发光染料在环介导等温扩增步骤之前加入。
成分 母液 用量(μL) 终浓度
核酸模板   2.0  
F3 10μM 1 0.2μM
B3 10μM 1 0.2μM
FIP 10μM 1 1.6μM
BIP 10μM 1 1.6μM
dNTP 10mM 3.5 1.4mM
MgSO 4 100mM 1.5 6mM
等温扩增缓冲液 10X 2.5 1X
Bst DNA聚合酶 8U/μL 1 0.32U/μL
聚集诱导发光染料   1  
双蒸水   补至25μL  
反应温度60-65℃，水浴加热，反应时间20-40min。
反应结束后，置于紫外灯下肉眼观察，反应体系发出蓝色荧光，为阳性，图中以灰度表示；不发出荧光为阴性。结果如图1所示，原图为带颜色的蓝色荧光照片，为了更清晰的呈现结果，右图为左图的反色，管1为阳性反应，管2为阴性反应。说明本发明的检测方法能够实现对新型冠状病毒的有效检测，特异性强。
实施例2 AIE染料快速扩增检验
为了验证本发明的检测方法的检测病毒的时间最短需要多久，进行了以下实验：
采用如下引物(5’-3’方向)：
外引物F3：GCCAAAAGGCTTCTACGCA；
外引物B3：TTTGGCCTTGTTGTTGTTGG；
内引物FIP：TCCCCTACTGCTGCCTGGAGGCAGTCAAGCCTCTTCTCG；
内引物BIP：TCCTGCTAGAATGGCTGGCAATTTTGCTCTCAAGCTGGTTCA。
分为11组，管1-10中加入新型冠状病毒的核酸作为模板，管11中不加入，以水代替，作为阴性对照。
反应体系如下(总体积为25μL)：
成分 母液 用量(μL) 终浓度
核酸模板   2.0  
F3 10μM 1 0.2μM
B3 10μM 1 0.2μM
FIP 10μM 1 1.6μM
BIP 10μM 1 1.6μM
dNTP 10mM 3.5 1.4mM
MgSO 4 100mM 1.5 6mM
等温扩增缓冲液 10X 2.5 1X
Bst DNA聚合酶 8U/μL 1 0.32U/μL
聚集诱导发光染料   1  
双蒸水   补至25μL  
在水浴锅里进行扩增，设置1min、2min、3min、4min、5min、10min、15min、20min、30min、40min十个时间梯度，温度64℃。
反应结束后，置于紫外灯下肉眼观察，结果如图2所示，原图为带颜色的蓝色荧光照片，图中以灰度表示，为了更清晰的呈现结果，下图为上图的反色，管1为64℃扩增1min，管2为64℃扩增2min，管3为64℃扩增3min，管4为64℃扩增4min，管5为64℃扩增5min，管6为64℃扩增10min，管7为64℃扩增15min，管8为64℃扩增20min，管9为64℃扩增30min，管10为64℃扩增40min，管11为阴性对照。结果显示由管7开始出现蓝色荧光，管7到管10荧光强度越来越强，说明本发明的检测 方法在64℃条件下，15min就能明显检测到病毒的目标基因，可以大大缩短检测的时间。
实施例3本发明检测方法的灵敏度检验
为了验证本发明的检测方法是否可以显著提高检测的灵敏度，能够检测到最低多少浓度的目的片段，开展以下实验。
以新冠病毒特异性基因序列nucleocapsid(N)为例，将新冠病毒按10倍倍比稀释，可视化LAMP检测方法反应体系和条件按下述进行。
采用如下引物(5’-3’方向)：
外引物F3：GCCAAAAGGCTTCTACGCA；
外引物B3：TTTGGCCTTGTTGTTGTTGG；
内引物FIP：TCCCCTACTGCTGCCTGGAGGCAGTCAAGCCTCTTCTCG；
内引物BIP：TCCTGCTAGAATGGCTGGCAATTTTGCTCTCAAGCTGGTTCA。
分为9组，管1-8中加入新型冠状病毒的核酸作为模板，管9中不加入，以水代替，作为阴性对照。
反应体系如下(总体积为25μL)：
成分 母液 用量(μL) 终浓度
核酸模板   2.0  
F3 10μM 1 0.2μM
B3 10μM 1 0.2μM
FIP 10μM 1 1.6μM
BIP 10μM 1 1.6μM
dNTP 10mM 3.5 1.4mM
MgSO 4 100mM 1.5 6mM
等温扩增缓冲液 10X 2.5 1X
Bst DNA聚合酶 8U/μL 1 0.32U/μL
聚集诱导发光染料   1  
双蒸水   补至25μL  
反应温度60-65℃，水浴加热，反应时间20-40min。
反应结束后，置于紫外灯下肉眼观察，结果如图3所示，原图为带颜色的蓝色荧光照片，为了更清晰的呈现结果，下图为上图的反色，管1为10 8拷贝数，管2为10 7拷贝数，管3为10 6拷贝数，管4为10 5拷贝数，管5为10 4拷贝数，管6为10 3拷贝数，管7为10 2拷贝数，管8为10拷贝数，管9为阴性对照。新冠病毒可视化LAMP检测方法的检测限为10拷贝数/管，在如此低的浓度下仍然可以检测到新冠病毒，说明本发明的检测方法灵敏度高。
实施例4大肠杆菌基因扩增检测
为了验证本发明的检测方法除了能够检测病毒，能否用于细菌的检测且有效，进行了以下实验。
通过查阅文献筛选出大肠杆菌中保守性高、特异性强的核酸序列作为靶序列，使用在线软件Primer Explorer v5设计出LAMP引物并合成，得到如下引物(5’-3’方向)：
外引物F3：CACCTTCATGGATATCGAGATT；
外引物B3：TGGAGGATTTAAGCCATCTC；
内引物FIP：CGAGCGTACAGCTGCAAAATGATATCTTTCGATACCACGACCT；
内引物BIP：CCCTTCTCCCTTTGTAACAAGATGACGCATAGTCAGCCCAT；
环引物LF：TAACGAAAGCCTGGGGCG；
环引物LB：CCTGTCATCGACAGCAACATTCA。
分为2组，管1中加入大肠杆菌的核酸作为模板，管2中不加入，以水代替，作为阴性对照。
反应体系如下(总体积为25μL)：
成分 母液 用量(μL) 终浓度
核酸模板   2.0  
F3 10μM 1 0.2μM
B3 10μM 1 0.2μM
FIP 10μM 1 1.6μM
BIP 10μM 1 1.6μM
LF 10μM 1 0.4μM
LB 10μM 1 0.4μM
dNTP 10mM 3.5 1.4mM
MgSO 4 100mM 1.5 6mM
等温扩增缓冲液 10X 2.5 1X
Bst DNA聚合酶 8U/μL 1 0.32U/μL
聚集诱导发光染料   1  
双蒸水   补至25μL  
反应温度60-65℃，水浴加热，反应时间20-40min。
反应结束后，置于紫外灯下肉眼观察，反应体系发出蓝色荧光，为阳性，图中以灰度表示；不发出荧光为阴性。结果如图1所示，原图为带颜色的蓝色荧光照片，为了更清晰的呈现结果，右图为左图的反色，管1为阳性反应，管2为阴性反应。说明本发明的检测方法能够实现对大肠杆菌的有效检测，特异性强。大肠杆菌的检测在畜牧业上具有很大的应用价值，是畜牧业中对于肉类品质鉴定的重要指标。
实施例5 AIE染料快速扩增大肠杆菌检验
为了验证本发明的检测方法的检测细菌的时间最短需要多久，进行了以下实验：
采用如下引物(5’-3’方向)：
外引物F3：CACCTTCATGGATATCGAGATT；
外引物B3：TGGAGGATTTAAGCCATCTC；
内引物FIP：CGAGCGTACAGCTGCAAAATGATATCTTTCGATACCACGACCT；
内引物BIP：CCCTTCTCCCTTTGTAACAAGATGACGCATAGTCAGCCCAT；
环引物LF：TAACGAAAGCCTGGGGCG；
环引物LB：CCTGTCATCGACAGCAACATTCA。
分为11组，管2-11中加入大肠杆菌的核酸作为模板，管1中不加入，以水代替，作为阴性对照。
反应体系如下(总体积为25μL)：
成分 母液 用量(μL) 终浓度
核酸模板   2.0  
F3 10μM 1 0.2μM
B3 10μM 1 0.2μM
FIP 10μM 1 1.6μM
BIP 10μM 1 1.6μM
LF 10μM 1 0.4μM
LB 10μM 1 0.4μM
dNTP 10mM 3.5 1.4mM
MgSO 4 100mM 1.5 6mM
等温扩增缓冲液 10X 2.5 1X
Bst DNA聚合酶 8U/μL 1 0.32U/μL
聚集诱导发光染料   1  
双蒸水   补至25μL  
在水浴锅里进行扩增，设置1min、2min、3min、4min、5min、10min、15min、20min、30min、40min十个时间梯度，温度64℃。
反应结束后，置于紫外灯下肉眼观察，结果如图5显示，原图为带颜色的蓝色荧光照片，为了更清晰的呈现结果，下图为上图的反色，管1为阴性对照，管2为64℃扩增1min，管3为64℃扩增2min，管4为64℃扩增3min，管5为64℃扩增4min，管6为64℃扩增5min，管7为64℃扩增10min，管8为64℃扩增15min，管9为64℃扩增20min，管10为64℃扩增30min，管11为64℃扩增40min。由图中可知，由管8开始出现蓝色荧光，图中以灰度表示，管8到管11荧光强度越来越强，说明本发明的检测方法在64℃条件下，15min就能明显检测到细菌的目标基因，可以大大缩短检测的时间。
实施例6金黄色葡萄球菌基因扩增检测
通过查阅文献筛选出金黄色葡萄球菌中保守性高、特异性强的核酸序列作为靶序列，使用在线软件Primer Explorer v5设计出LAMP引物并合成，得到如下引物(5’-3’方向)：
外引物F3：GACGATTGTTTGCTTGTTCT；
外引物B3：CCAAGAGATAGCCCCTAGA；
内引物FIP：GAGTGTGAATAAATATGGCTGTTCGTAGCAGCAATGCATCCAA；
内引物BIP：AATGGTATTAGTCGCATTACCCGAGCAGAAGAAATTCTAAAACTCA；环引物LF：AAAGTTACTGTGTTTGGACCTG；
环引物LB：CACTTATTTTAGCTATATTCAATAA。
分为2组，管1中加入金黄色葡萄球菌核酸作为模板，管2中不加入，以水代替，作为阴性对照。
反应体系如下(总体积为25μL)：
成分 母液 用量(μL) 终浓度
核酸模板   2.0  
F3 10μM 1 0.2μM
B3 10μM 1 0.2μM
FIP 10μM 1 1.6μM
BIP 10μM 1 1.6μM
LF 10μM 1 0.4μM
LB 10μM 1 0.4μM
dNTP 10mM 3.5 1.4mM
MgSO 4 100mM 1.5 6mM
等温扩增缓冲液 10X 2.5 1X
Bst DNA聚合酶 8U/μL 1 0.32U/μL
聚集诱导发光染料   1  
双蒸水   补至25μL  
反应温度60-65℃，水浴加热，反应时间20-40min。
反应结束后，置于紫外灯下肉眼观察，反应体系发出蓝色荧光，为阳性，图中以灰度表示；不发出荧光为阴性。结果如图6所示，原图为带颜色的蓝色荧光照片，为了更清晰的呈现结果，右图为左图的反色，管1为阳性反应，管2为阴性反应。说明本发明的检测方法能够实现对金黄色葡萄球菌的有效检测，特异性强。
实施例7志贺菌基因扩增检测
通过查阅文献筛选出志贺菌中保守性高、特异性强的核酸序列作为靶序列，使用在线软件Primer Explorer v5设计出LAMP引物并合成，得到如下引物(5’-3’方向)：
外引物F3：ATACCGTCTCTGCACGCA；
外引物B3：GCCTTCTGATGCCTGATGG；
内引物FIP：CTGTCGAAGCTCCGCAGAGGGGATTCCGTGAACAGGTCG；
内引物BIP：AGTCTTTCGCTGCTGCTGCTGATCCGGAGATTGTTCCATGTG；
环引物LF：GAGTTTTTCCAGCCATGCAG；
环引物LB：TGCCACTGAGAGCTGTGAG。
分为2组，管1中加入志贺菌核酸作为模板，管2中不加入，以水代替，作为阴性对照。
反应体系如下(总体积为25μL)：
成分 母液 用量(μL) 终浓度
核酸模板   2.0  
F3 10μM 1 0.2μM
B3 10μM 1 0.2μM
FIP 10μM 1 1.6μM
BIP 10μM 1 1.6μM
LF 10μM 1 0.4μM
LB 10μM 1 0.4μM
dNTP 10mM 3.5 1.4mM
MgSO 4 100mM 1.5 6mM
等温扩增缓冲液 10X 2.5 1X
Bst DNA聚合酶 8U/μL 1 0.32U/μL
聚集诱导发光染料   1  
双蒸水   补至25μL  
反应温度60-65℃，水浴加热，反应时间20-40min。
反应结束后，置于紫外灯下肉眼观察，反应体系发出蓝色荧光，为阳性，图中以灰度表示；不发出荧光为阴性。结果如图7所示，原图为带颜色的蓝色荧光照片，为了更清晰的呈现结果，右图为左图的反色，管1为阳性反应，管2为阴性反应。说明本发明的检测方法能够实现对志贺菌的有效检测，特异性强。
实施例8人乳头瘤病毒基因扩增检测
通过查阅文献筛选出人乳头瘤病毒16亚型中保守性高、特异性强的核酸序列作为靶序列，使用在线软件Primer Explorer v5设计出LAMP引物并合成，得到如下引物(5’-3’方向)：
外引物F3：GCACCAAAAGAGAACTGC；
外引物B3：AGCATATGGATTCCCATCTC；
内引物FIP：TTGTTTGCAGCTGGGGAATTTATTTCAGGACCCAACAGGAG；
内引物BIP：TTAGAATGTGTGTACTGCAAGCATCCCGAAAAGCAAAGTCA；
环引物LF：GGTAACTTTCTGGGTCGCTCC；
环引物LB：AGTTACTGCGACGTGAGGT。
分为2组，管1中加入人乳头瘤病毒核酸作为模板，管2中不加入，以水代替，作为阴性对照。
反应体系如下(总体积为25μL)：
成分 母液 用量(μL) 终浓度
核酸模板   2.0  
F3 10μM 1 0.2μM
B3 10μM 1 0.2μM
FIP 10μM 1 1.6μM
BIP 10μM 1 1.6μM
LF 10μM 1 0.4μM
LB 10μM 1 0.4μM
dNTP 10mM 3.5 1.4mM
MgSO 4 100mM 1.5 6mM
等温扩增缓冲液 10X 2.5 1X
Bst DNA聚合酶 8U/μL 1 0.32U/μL
聚集诱导发光染料   1  
双蒸水   补至25μL  
反应温度60-65℃，水浴加热，反应时间20-40min。
反应结束后，置于紫外灯下肉眼观察，反应体系发出蓝色荧光，为阳性，图中以灰度表示；不发出荧光为阴性。结果如图8所示，原图为带颜色的蓝色荧光照片，为了更清晰的呈现结果，右图为左图的反色，管1为阳性反应，管2为阴性反应。说明本发明的检测方法能够实现对人乳头瘤病毒的有效检测，特异性强。
实施例9转基因扩增检测
本发明的检测方法不仅可以用于细菌和病毒等病原微生物的检测，还可以用来检测动植物中是否转入了基因。操作方法与细菌和病毒的检测相似，只是把引物片段替换为靶向转基因动植物中特定的核酸序列的引物，提取待测的动植物的基因组DNA，利用前述实施例的操作步骤和检测方法，如果在紫外灯下观察有荧光，代表待测的动植物中转入了基因，是转基因的动植物，如果没有荧光信号，则非转基因动植物。
综上，本发明公开了一种荧光可视化的环介导等温扩增(LAMP)产物检测方法及其试剂盒和应用，本发明的检测方法在环介导等温扩增体系中添加了聚集诱导发光染料(AIE)，反应结束后，通过紫外灯观察，判断反应结果。本发明的试剂盒在LAMP试剂盒的基础上添加聚集诱导发光染料，提高了检测的灵敏度，检测限为10拷贝数/管，还能够缩短检测的时间，最少只需15分钟就能明显检测到目标基因。本发明的检测方法无需后续的电泳检测，在紫外灯下反应体系发出蓝色荧光为阳性，颜色不发光为阴性，实现了荧光可视化，结果判定直观、准确，简单明了。聚集诱导发光染料提前加入到LAMP反应体系中，克服了气溶胶污染的问题，实现了不开盖检测，可快速、精确的检测病原微生物和转基因动植物。
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种荧光可视化的环介导等温扩增产物的检测方法，其特征在于，在环介导等温扩增体系中添加聚集诱导发光染料，反应结束后，通过紫外灯观察，判断反应结果。
  2. 根据权利要求1所述的检测方法，其特征在于，所述反应的温度为60-65℃，所述反应的时间为15-40min。
  3. 根据权利要求1所述的检测方法，其特征在于，所述的聚集诱导发光染料在环介导等温扩增步骤之前加入。
  4. 一种荧光可视化的环介导等温扩增产物检测试剂盒，其特征在于，包括如下成份：
    dNTP、MgSO 4、等温扩增缓冲液、Bst DNA聚合酶、聚集诱导发光染料、双蒸水。
  5. 一种新冠病毒检测试剂盒，其特征在于，包括如下成份：
    dNTP、MgSO 4、等温扩增缓冲液、Bst DNA聚合酶、聚集诱导发光染料、双蒸水、引物；
    所述引物包含4个，其序列5’-3’方向分别为GCCAAAAGGCTTCTACGCA、TTTGGCCTTGTTGTTGTTGG、TCCCCTACTGCTGCCTGGAGGCAGTCAAGCCTCTTCTCG、TCCTGCTAGAATGGCTGGCAATTTTGCTCTCAAGCTGGTTCA。
  6. 一种大肠杆菌检测试剂盒，其特征在于，包括如下成份：
    dNTP、MgSO 4、等温扩增缓冲液、Bst DNA聚合酶、聚集诱导发光染料、双蒸水、引物；
    所述引物包含6个，其序列5’-3’方向分别为CACCTTCATGGATATCGAGATT、TGGAGGATTTAAGCCATCTC、CGAGCGTACAGCTGCAAAATGATATCTTTCGATACCACGACCT、 CCCTTCTCCCTTTGTAACAAGATGACGCATAGTCAGCCCAT、TAACGAAAGCCTGGGGCG、CCTGTCATCGACAGCAACATTCA。
  7. 一种金黄色葡萄球菌检测试剂盒，其特征在于，包括如下成份：
    dNTP、MgSO 4、等温扩增缓冲液、Bst DNA聚合酶、聚集诱导发光染料、双蒸水、引物；
    所述引物包含6个，其序列5’-3’方向分别为GACGATTGTTTGCTTGTTCT、CCAAGAGATAGCCCCTAGA、GAGTGTGAATAAATATGGCTGTTCGTAGCAGCAATGCATCCAA、AATGGTATTAGTCGCATTACCCGAGCAGAAGAAATTCTAAAACTCA、AAAGTTACTGTGTTTGGACCTG、CACTTATTTTAGCTATATTCAATAA。
  8. 一种志贺菌检测试剂盒，其特征在于，包括如下成份：
    dNTP、MgSO 4、等温扩增缓冲液、Bst DNA聚合酶、聚集诱导发光染料、双蒸水、引物；
    所述引物包含6个，其序列5’-3’方向分别为ATACCGTCTCTGCACGCA、GCCTTCTGATGCCTGATGG、CTGTCGAAGCTCCGCAGAGGGGATTCCGTGAACAGGTCG、AGTCTTTCGCTGCTGCTGCTGATCCGGAGATTGTTCCATGTG、GAGTTTTTCCAGCCATGCAG、TGCCACTGAGAGCTGTGAG。
  9. 一种人乳头瘤病毒检测试剂盒，其特征在于，包括如下成份：
    dNTP、MgSO 4、等温扩增缓冲液、Bst DNA聚合酶、聚集诱导发光染料、双蒸水、引物；
    所述引物包含6个，其序列5’-3’方向分别为GCACCAAAAGAGAACTGC、AGCATATGGATTCCCATCTC、TTGTTTGCAGCTGGGGAATTTATTTCAGGACCCAACAGGAG、 TTAGAATGTGTGTACTGCAAGCATCCCGAAAAGCAAAGTCA、GGTAACTTTCTGGGTCGCTCC、AGTTACTGCGACGTGAGGT。
  10. 权利要求1所述的检测方法在病原微生物检测和转基因检测方面的应用，所述病原微生物包括病毒、细菌。
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