Patent Publication Number: US-2022213545-A1

Title: Kit for aflatoxin b1 (afb1) monitoring

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     The present disclosure claims the priority to the Chinese patent application with the filing number 202110015995.9, filed with the Chinese Patent Office on Jan. 7, 2021, and entitled “A kit for aflatoxin B1 (AFB1) monitoring, preparation and detection method thereof”, which is incorporated herein by reference in its entirety. 
     TECHNICAL FIELD 
     The invention displays an aflatoxin B 1  (AFB 1 ) detection kit and a AFB 1  detection method. The invention belongs to the technical field of detecting harmful substances. 
     BACKGROUND ART 
     AFB 1  is low-molecular-weight natural secondary metabolites produced by  Aspergillus flavus  and  Aspergillus parasiticus . AFB 1  is the most toxic among all mycotoxins, posing teratogenic, carcinogenic, and mutagenic risks to humans, and has been labeled as a group I carcinogen in humans by the International Agency for Research on Cancer (IARC). Given the serious impact of this mycotoxin on human health, governments and relevant organizations have formulated strict regulations and testing methods to limit the level of aflatoxin in food. 
     Traditionally, the most widely used mycotoxin analysis techniques include high performance liquid chromatography (HPLC), gas chromatography/mass spectrometry (GC/MS) and probe-based enzyme-linked immunoassay (ELISA). Instrumental analysis methods need high cost instruments and equipment, long test times, and skilled lab researchers for the detection process. In addition, traditional ELISA requires time-consuming culture and specific elution conditions, and the enzymes involved in the experiment are prone to denaturation or inactivation. Therefore, it is urgent to carry out in-depth research in this field and establish a rapid and effective method for the separation and detection of AFB 1  in food. 
     Aptamers are short, single-stranded oligonucleotide sequences (DNA, RNA, or nucleic acid analogs) selected from a nucleic acid molecular library using the in vitro systematic evolution of ligands by exponential enrichment (SELEX) method. Like antibodies, aptamers have strict recognition and high affinity for binding ligands. Because of its advantages of easy synthesis, easy storage and easy modification, it has a wide application prospect in analysis and detection. 
     DNA nanostructures are assembled from single strand DNA through complementary base pairs, which have high controllability, biocompatibility and stability. In recent years, DNA nanostructures have been increasingly used in the construction of biosensors. DNA nanostructures have many functions, such as drug delivery, signal carrying, signal amplification and rigid support, which greatly improve the performance of biosensors. 
     SUMMARY 
     In view of the problems in the prior art, the purpose of the present invention is to provide a kit for detecting AFB 1  and a method for detecting AFB 1 . The kit of the invention realizes dual signal amplification based on DNA Walker structure and hyperbranched fluorescent nanotree structure. This method not only enhances the detection signal of AFB 1  and improves the reaction speed, but also realizes the high sensitive and rapid detection of AFB 1 . 
     In order to achieve the above purpose, the invention adopts the following technical scheme: 
     A detection kit for AFB 1 , which contains DNA walker structure, endonuclease, hairpin H1 and H2. 
     The DNA Walker structure is gold nanoparticles (AuNPs) modified with WA double strand and DNA tetrahedrons (DTNs). 
     The WA is a double stranded structure composed of aptamer A of AFB 1  and its partially complementary nucleic acid sequence W. 
     The sequence E1 on the W chain and the sequence E2 on the S1 chain can form the recognition site of endonuclease by base complementary pairing. 
     There is a 4-15 base sequence E1 in the nucleic acid sequence W and A are not complementary. The nucleic acid sequence at the junction of DTNs and gold nanoparticles contains a 4-15 base sequence E2. The sequence E1 on the W chain and the sequence E2 on the S1 chain can form the recognition site of endonuclease by base complementary pairing. 
     Both ends of the hairpin structure H1 are respectively modified with a fluorescent group and a fluorescence quenching group. 
     The DTNs are self-assembled by four DNA single strands through base complementary. The nucleic acid sequence of DTNs pivot is complementary to the partial sequence of hairpin structure H1, which is used to open the hairpin structure of H1. The hairpin structure H1 is complementary to the partial sequence base of hairpin structure H2, which is used to open the hairpin structure of H2. 
     On the basis of the above scheme, the cutting endonuclease is Nt.BsmAI. 
     On the basis of the above scheme, the sequence of aptamer A of AFB 1  is shown in SEQ ID NO:1. 
     On the basis of the above scheme, the nucleic acid sequence of W is shown in sequence ID NO:2. The 5 ′end of W is modified by sulfhydryl group. 
     On the basis of the above scheme, the four DNA single stranded sequences of the DTNs are shown in the sequence of SEQ ID NO:3-6. The 5′ end of the sequence ID NO:3 is modified by sulfhydryl group. 
     On the basis of the above scheme, the nucleic acid sequence of the H1 is shown in SEQ ID NO:7. 
     On the basis of the above scheme, the fluorescent group in the H1 is FAM fluorescent group, and the fluorescence quenching group is Dabcyl. 
     On the basis of the above scheme, the nucleic acid sequence of the H2 is shown in SEQ ID NO:8. 
     On the basis of the above scheme, DNA Walker structure is prepared in the following steps: 
     Thiol groups of WA and DTNs were reduced by TCEP for 30 min. The activated WA and DTNs were mixed in a molar ratio of 1:4, and then 0.1% AuNPs solution was added to the mixture at 4° C. overnight. Next, 1M sodium chloride solution was added to the above solution every 1 h for a total of 5 times to ensure that the final concentration of sodium chloride is 0.15 M. After each addition of sodium chloride, the solution needs to be sonicated for 10 s. Finally, the uncoupled WA and DTNs were removed by centrifugation to obtain the DNA walker structure. 
     The steps for using the above kit to detect AFB 1  are as follows: 
     (1) The test sample solution and DNA walker solution were mixed at 37° C. and incubated for 0.5 h. Then the cutting endonuclease Nt.BsmAI was added under 37° C. for 0.5 h. Then the mixture was kept at 65° C. for 20 min. 
     (2) Adding sodium chloride solution to the solution after reaction in step (1), the AuNPs was precipitated in the salt solution to retain the supernatant. 
     (3) H1 and H2 were added to above solution (2) for the reaction at 37° C. for 15 min. The molar ratio of H1 and H2 was 1:1. 
     (4) The fluorescence intensity of the solution after reaction in step (3) was detected at 490 nm excitation wavelength. The measured fluorescence intensity was introduced into the standard curve. 
     When the kit of the invention detects AFB 1  in the sample, the adopted sample solution needs to be a clear and transparent liquid. 
     The advantages of the technical scheme of the invention are as follows: 
     The DNA Walker structure of the invention is a signal amplification structure mediated by the target AFB 1 . DTNs not only concatenate the DNA Walker structure and the hyperbranched fluorescent nanotree, but also provide multiple vertex for hyperbranched fluorescent nanotree. The invention accelerates the reaction speed and realize the dual amplification of signal. 
     The method of the invention has the characteristics of low detection cost, fast detection and low requirements for detection instruments. 
     In particular, the invention first uses DNA Walker structure, DNA tetrahedron structure and hyperbranched fluorescent nanotree as signal amplification means to detect mycotoxins. The invention can improve the sensitivity of aptasensor and provide a new technology for rapid screening of mycotoxin contamination in food. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIG. 1 : Schematic diagram of detection principle of the method of the invention. 
         FIG. 2 : The sensitivity detection of the detection kit. 
         FIG. 3 : The specificity detection of the detection kit. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Unless otherwise specified, the terms used in the description of the invention typically have the meanings commonly appreciated by those ordinarily skilled in the art. 
     The invention is further detailed below in combination with embodiments and reference data. The following embodiments are only used for illustratively explaining the invention, and are not intended to limit the scope of the invention in any form. 
     Embodiment 1 
     The detection principle of the invention is as follows: 
     DTNs are formed by complementary self-assembly of S1, S2, S3 and S4. There is a single strand extension sequence at the four vertices of DTNs. WA double stranded structure composed of aptamer A of AFB 1  and its partially complementary nucleic acid sequence W. The 5 ′end of S1 and W chain were modified by sulfhydryl group. After thiol reduction by TCEP reductant, DTNs and WA were modified on the surface of AuNPs to form DNA Walker structure. 
     In the presence of AFB 1 , aptamer A combined with AFB 1  and dissociated from AuNPs with DNA Walker structure. Subsequently, the W chain was in a single chain state and began to swim on the surface of AuNPs driven by base complementation. The binding of E1 on W chain with E2 on S1 forms the recognition site of endonuclease, which triggers the cleavage of S1 chain by endonuclease and makes DTN dissociate from the surface of AuNPs. W would swim to the next binding site until the DTNs are cut down to complete the first amplification of the signal. 
     The nucleic acid sequence of DTNs is complementary to the partial sequence of hairpin H1, resulting in the opening of hairpin H1. The extended sequence of H1 is complementary to the partial sequence of H2, resulting in the opening of the hairpin structure of H2. These processes lead to chain reaction and further construct hyperbranched nanostructures. Both ends of the hairpin structure H1 are respectively modified with a fluorescent group and a fluorescence quenching group. In the open H1, the fluorescence group and the fluorescence quenching group are separated, so that the fluorescence is restored. With the continuous opening of H1 and H2, the fluorescence signal brought by FAM is also expanding, completing the second signal amplification. AFB 1  was determined by fluorescence intensity. 
     A detection kit for AFB 1 , which contains DNA walker structure, endonuclease, hairpin H1 and H2. 
     The DNA walker structure is prepared by the following method: 
     (1) Self-Assembly of DTNs 
     The four ssDNAs were mixed equivalently in buffer (10 mM Tris-HCl, 2.5 mM MgCl2, 100 mM NaCl pH 8.0), and the mixture was heated at 95° C. for 5 min, 45° C. for 30 min. Finally, the assembled DTNs were purified by 3% agarose gel electrophoresis. 
     (2) Hybridization Between W and A 
     The W and A were mixed equivalently, and the mixture was heated at 95° C. for 5 min and then slowly cooled to 25° C. 
     (3) Assembly of DNA Walker Structure 
     Thiol groups of WA and DTNs were reduced by TCEP for 30 min. The activated WA and DTNs were mixed in a molar ratio of 1:4, and then 0.1% AuNPs solution was added to the mixture at 4° C. overnight. Next, 1M sodium chloride solution was added to the above solution every 1 h for a total of 5 times to ensure that the final concentration of sodium chloride is 0.15 M. After each addition of sodium chloride, the solution needs to be sonicated for 10 s. Finally, the uncoupled WA and DTNs are removed by centrifugation to obtain the DNA walker structure. 
     The above sequence is shown in the following table: 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                   
                 Name 
                 Sequence (5′-3′) 
                 Number 
               
               
                   
               
             
            
               
                   
                 A 
                   GTTGGGCACGTGTTGTCT CTCTGTGTCTCG 
                 SEQ ID 
               
               
                   
                   
                 TGCCCTTCGCTAGGCCC 
                 NO: 1 
               
               
                   
               
               
                   
                 W 
                 SH-TTTTTTTTTTTTTTTTTTTTT AGACAA   
                 SEQ ID 
               
               
                   
                   
                 
                   CACGTGCCCAAC 
                   GGAGAC 
                 
                 NO: 2 
               
               
                   
               
               
                   
                 S1 
                 SH- GTCTCC *GTTTCAAGCGCAGCACTTAC 
                 SEQ ID 
               
               
                   
                   
                 CTGTATCCTTTCCGAGTTACGTCTGTCCCT 
                 NO: 3 
               
               
                   
                   
                 AGAGTTTTCCTACTTACAAGAGCCGGATAC 
                   
               
               
                   
                   
                 GC 
                   
               
               
                   
               
               
                   
                 S2 
                   TCAGTCTAGGATTCGGCGTGGGTT TTTGGA 
                 SEQ ID 
               
               
                   
                   
                 TACAGGTAAGTGCTGCGCTTGTTTAATGGA 
                 NO: 4 
               
               
                   
                   
                 ACTTGAGATGTTAGGGAGTTTTCTTAGCTA 
                   
               
               
                   
                   
                 GGTGTGATACATTAC 
                   
               
               
                   
               
               
                   
                 S3 
                   TCAGTCTAGGATTCGGCGTGGGTT TTTTAT 
                 SEQ ID 
               
               
                   
                   
                 CACCAGGCAGTTGACAGTGTATTTCTCCCT 
                 NO: 5 
               
               
                   
                   
                 AACATCTCAAGTTCCATTTTTGCGTATCCG 
                   
               
               
                   
                   
                 GCTCTTGTAAGTAGG 
                   
               
               
                   
               
               
                   
                 S4 
                   TCAGTCTAGGATTCGGCGTGGGTT TTTTAC 
                 SEQ ID 
               
               
                   
                   
                 ACTGTCAACTGCCTGGTGATATTTACTCTA 
                 NO: 6 
               
               
                   
                   
                 GGGACAGACGTAACTCGGTTTGTAATGTAT 
                   
               
               
                   
                   
                 CACACCTAGCTAAGA 
                   
               
               
                   
               
               
                   
                 H1 
                 FAM-GCGTGGGTTGCGCTGATCAAGACTCC 
                 SEQ ID 
               
               
                   
                   
                 ATGA AACCCACGCCGAATCCTAGACTGA GC 
                 NO: 7 
               
               
                   
                   
                 GCTG-Dabcyl 
                   
               
               
                   
               
               
                   
                 H2 
                 TCATGGAGTCTTGATCAGCGCAACCCACGA 
                 SEQ ID 
               
               
                   
                   
                 CAGCGC TCAGTCTAGGATTCGGCGTGGGTT   
                 NO: 8 
               
               
                   
               
            
           
         
       
     
     The sequence of single underline in the above table is the complementary sequence of A and W. The double underlined sequences are S2, S3, S4 and the complementary sequences of H1 and H2. The bold sequence represents E1 on the w Chain and E2 on the S1 chain. * is the cleavage site of endonuclease. 
     Embodiment 2 
     Method for detecting AFB 1  using the kit of embodiment 1: 
     (1) The sample solution was filtered and diluted 10 times. The 10 μL test sample solution and DNA walker solution were mixed at 37° C. and incubated for 0.5 h. Then the cutting endonuclease Nt.BsmAI was added under 37° C. for 0.5 h. The mixture was kept at 65° C. for 20 min. 
     (2) Adding sodium chloride solution to the solution after reaction in step (1), the AuNPs is precipitated in the salt solution to retain the supernatant. 
     (3) H1 and H2 were added to above solution (2) for the reaction at 37° C. for 15 min. The molar ratio of H1 and H2 was 1:1. 
     (4) The fluorescence intensity of the solution after reaction in step (3) was detected at 490 nm excitation wavelength. The measured fluorescence intensity was introduced into the standard curve. 
     Sensitivity of the Kit of Embodiment 1 
     Different concentrations of AFB 1  were used to test the sensitivity of the test kit in embodiment 1 of the present invention by the test method in embodiment 2. The AFB 1  of different concentration gradients were 1, 2, 5, 10, 20, 50, 100, 200, 500 pg/mL. As shown in  FIG. 2 , the relationship between AFB 1  concentration and fluorescence intensity was y=253.4 ln(x)−67.007, R 2 =0.9912. The detection range of embodiment 1 kit was 1-500 pg/mL, and the detection limit was 0.5 pg/mL. 
     The Specificity Detection of the Kit of Embodiment 1 
     The specificity of the kit was further checked using other possible interfering mycotoxins, such as aflatoxin M1 (AFM 1 ), zearalenone (ZEN) and ochratoxin A (OTA). The concentration of each toxin was 100 pg/mL. The specific results are shown in  FIG. 3 . The value of AFB 1  is significantly higher than that of other mycotoxins. Therefore, the kit of embodiment 1 of the invention has high specificity for AFB 1 . 
     The embodiments are only preferred ones of the invention, and are not intended to limit the invention in any form. Any skilled in the art can transform or modify the technical contents disclosed below to obtain equivalent embodiments. Any simple modifications or equivalent transformations to the following embodiments according to the technical essence of the invention without deviating from the contents of the technical solutions of the invention should also fall within the protection scope of the technical solutions of the invention.