WO2020114322A1 - 一种用来鉴定评价黄曲霉毒素产毒菌株产毒力的方法 - Google Patents

一种用来鉴定评价黄曲霉毒素产毒菌株产毒力的方法 Download PDF

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WO2020114322A1
WO2020114322A1 PCT/CN2019/121785 CN2019121785W WO2020114322A1 WO 2020114322 A1 WO2020114322 A1 WO 2020114322A1 CN 2019121785 W CN2019121785 W CN 2019121785W WO 2020114322 A1 WO2020114322 A1 WO 2020114322A1
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aflatoxin
strain
virulence
gene
pcr
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张奇
李培武
白艺珍
李慧
姜俊
张文
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中国农业科学院油料作物研究所
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Priority to JP2020552711A priority patent/JP7122022B2/ja
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Definitions

  • the invention belongs to the biological field, and in particular relates to a method for identifying and evaluating the virulence of aflatoxin-producing strains.
  • Aflatoxin is by far the most toxic class of mycotoxins found. Taking aflatoxin B1 as an example, its toxicity is 10 times that of potassium cyanide and 68 times that of arsenic. It is classified as a class I carcinogen by the International Cancer Organization. Aflatoxin can easily contaminate grain, oil and other crop products such as peanuts, corn and rice, as well as many plant products such as walnuts, pistachios and Chinese herbal medicines. There have been many cases of human and animal poisoning caused by aflatoxin at home and abroad. According to the latest report from the International Agency for Research on Cancer (IARC), about 500 million people in developing countries alone are at risk of exposure to aflatoxin. my country is a region heavily polluted by aflatoxin.
  • IARC International Agency for Research on Cancer
  • Aflatoxin is mainly produced by fungi such as Aspergillus flavus and Aspergillus parasite. Studies have shown that the ability of different aflatoxin strains to produce aflatoxin-that is, the virulence can be hundreds of times different, strong strains are an important source of high pollution, but so far lack of specificity to quickly identify strong strains Effective method. At present, there are two main methods to identify the virulence of aflatoxin strains. One is to directly evaluate the aflatoxin production capacity of the strain after culturing the strain for a certain period of time.
  • This kind of method is long, it must first isolate the strain, then culture, and finally evaluate by detecting the content of aflatoxin; second, the factors affecting the biosynthesis of aflatoxin are very many and complex, the same strain is cultivated in different culture batches There is a large difference in the production of aflatoxin between the two, and the result is difficult to characterize the strain's own toxin production characteristics that should be constant, thereby affecting the accuracy and reliability of the method's identification evaluation results.
  • Another method is to evaluate the virulence of the strain by measuring the transcription level of the gene related to toxin production. There are reports in the literature to detect the Nor-1 gene to evaluate the virulence.
  • the present invention addresses the deficiencies of the prior art, and aims to provide a method for identifying and evaluating the virulence of aflatoxin-producing strains.
  • a method for identifying and evaluating the virulence of aflatoxin-producing strains measuring the yield of aflatoxin and Nor-1 gene transcription, and obtaining the ratio of aflatoxin production to Nor-1 gene transcription, according to the production of aflatoxin Identification of the ratio of the transcription amount with Nor-1 gene to evaluate the toxin production capacity of aflatoxin-producing strains.
  • the method for measuring the yield of aflatoxin is: culturing the strain of aflatoxin, taking the spores of the aflatoxin for shaking culture, and after the cultivation is completed, filtering the filtrate to determine the concentration of aflatoxin in the filtrate.
  • the medium used for cultivating the strain of Aspergillus flavus is CDA medium, and the culture conditions are: culture at 28°C and 90% humidity for 10 days;
  • the medium used for shaking culture of the spores of Aspergillus flavus is potato glucose liquid medium; culture conditions: shaking culture at 28°C and 200 rpm for 96 hours;
  • the standard method of immunoaffinity purification-high performance liquid chromatography was used to determine the concentration of aflatoxin in the filtrate of the spores of the aflatoxin after shaking culture.
  • the method for determining the transcription amount of Nor-1 gene cultivating the A. flavus strain, taking the spores of A. flavus for shaking culture, after the cultivation is completed, filtering and taking the mycelium of A. flavus, and drying to obtain dried cells Measure the amount of Nor-1 gene transcript in dried bacteria using conventional Nor-1 gene transcript measurement method.
  • the aflatoxin production and Nor-1 gene transcription can also be obtained by synchronous detection RT-PCR method, the specific steps include:
  • the phage displaying aflatoxin anti-idiotypic nanobody on the surface is phage VHH2-5, and the aflatoxin monoclonal antibody is aflatoxin monoclonal antibody 1C11.
  • the final concentrations of the upstream and downstream primers: Ph-F, Ph-R, Tq-nor1-F, and Tq-nor1-R are all 300-400 nM; Fluorescent probe: Ph-probe and Tq-probe final concentration 200 ⁇ 400nM; DNA polymerase final dosage: 0.5U ⁇ 1.0U; MgCl 2 final concentration: 1mM ⁇ 2mM; dNTPs final concentration: 200uM ⁇ 400uM.
  • the upstream primer Ph-F the nucleotide sequence is shown in SEQ ID NO.1; the downstream primer Ph-R, the nucleotide sequence is shown in SEQ ID NO.2; the fluorescent probe Ph-probe , The nucleotide sequence is shown in SEQ ID NO.3; the upstream primer Tq-nor1-F, the nucleotide sequence is shown in SEQ ID NO.4; the downstream primer Tq-nor1-R, the nucleotide sequence is shown in SEQ ID NO.5; fluorescent probe Tq-probe, nucleotide sequence shown in SEQ ID NO.6.
  • the reaction system of the synchronous RT-PCR amplification reaction includes: general purpose probe qPCR premix 5 ⁇ L, Ph-F 0.1 ⁇ L, Ph-R 0.1 ⁇ L, Ph-probe 0.1 ⁇ L, phage template 2 ⁇ L, Tq -nor1-F 0.1 ⁇ L, Tq-nor1-R 0.1 ⁇ L, Tq-probe 0.1 ⁇ L, Nor-1 gene template 1 ⁇ L, DNA polymerase 0.2 ⁇ L, MgCl 2 0.8 ⁇ L, dNTPs 0.2 ⁇ L, add H 2 O to make up 10 ⁇ L .
  • the conditions of the RT-PCR simultaneous amplification reaction are 95° C., 5 min; 95° C., 10 s, 60° C. 30 s, 40 cycles.
  • the concentration range of aflatoxin in the quantitative S-type standard curve of aflatoxin is 33.33ng/mL ⁇ 1.69pg/mL, and the minimum detection line LOD of aflatoxin is 0.018ng/mL; the nor- 1 The nor-1 gene copy number range in the quantitative standard curve of gene transcription quantity is 10 2 ⁇ 10 8 .
  • the virulence of the aflatoxin-producing strain is defined as Y
  • the ratio of aflatoxin production to Nor-1 gene transcription amount (AFT/Nor-1) is defined as X
  • the aflatoxin strain produces toxin
  • the present invention establishes a synchronous RT-PCR detection method for synchronously detecting the toxin production of Aspergillus flavus strain and nor-1 gene transcription amount.
  • AFT/Nor-1 produced by Aspergillus flavus strains
  • the ratio of toxic amount/nor-1 gene transcription amount is reliable and accurate, and can be used as an identification indicator to determine the virulence of Aspergillus flavus strains;
  • the amount of reagents is less, the cost is lower, and high-throughput detection can be achieved;
  • the synchronous detection RT -The PCR method simplifies the analysis mode, optimizes the experimental process and structure, and provides a detection platform and theoretical basis for the simultaneous analysis of aflatoxins and other small molecular substances in their synthetic pathways.
  • Figure 1 shows the concentration of DNA polymerase, dNTPs and MgCl 2 in optimized simultaneous RT-PCR reaction.
  • Figure 2 shows the evaluation of simultaneous RT-PCR amplification efficiency.
  • Figure 3 is a quantitative standard curve for quantification of aflatoxin B1 and nor-1 gene transcription by simultaneous RT-PCR.
  • Figure 4 is the quantitative of simultaneous RT-PCR: cross-reactivity with aflatoxins B1, B2, G1, G2, ZEN, DON, FB1.
  • Figure 5 shows the comparison of quantitative results of synchronous RT-PCR with HPLC and Nanodrop.
  • Figure 6 shows the correlation between aflatoxin production and Nor-1 gene expression (A), and the relationship between aflatoxin production and AFT/Nor-1 ratio (B).
  • the spore solution of Aspergillus flavus was shaken uniformly with a vortex shaker, and the spore solution of Aspergillus flavus was counted microscopically with a microscope.
  • the standard method of immunoaffinity purification-high performance liquid chromatography was used to determine the concentration of aflatoxin in the filtrate (preserved for future use) obtained by shaking culture of aflatoxin spores, and the conventional Nor-1 gene transcription relative amount determination method was used to determine The relative amount of Nor-1 gene transcription in the above-mentioned preserved dry bacterial cells.
  • the phage VHH 2-5 displaying aflatoxin anti-idiotype Nanobodies on the surface was developed by the Quality Inspection Center of the Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences and has been published in the journal literature “Yanru Wang; Peiwu Li; Zuzana Majkova; Candace RSBever; Hee Joo Kim; Qi Zhang; Julie E. Dechant; Shirley J. Gee; Bruce D. Hammock; Isolation of Alpaca Anti-Idiotypic Heavy-Chain Single-Domain Antibody for the Aflatoxin Immunoassay; Reported by Analytical 8298, 2013, 2013,
  • the bacteriophage used in this example was pre-stored in E. coli ER2738 in the laboratory and was obtained by amplification.
  • the amplification method is as follows:
  • the method for obtaining the Tq-nor1 DNA fragment of the nor-1 gene place 15 mL of potato dextrose liquid medium in a 50 mL Erlenmeyer flask, autoclave at 121° C. for 30 min, and add spore fluid of Aspergillus flavus N73 to a final concentration of 1 ⁇ 10 5 ml -1 , after shaking culture at 28°C and 200 rpm for 96h; squeeze the mycelium culture liquid out of the double-layer filter paper, dry at 65°C for 12h, quick freeze in liquid nitrogen, and grind to powder; accurately weigh 200mg of mycelial powder , DNA extraction kit (DNeasy Plant Mini Kit) was used to extract the genomic DNA of strain N73 according to the kit instructions, the nor-1 gene fragment product with a size of 400bp was amplified with the following primers, and extracted with EZNATM (OMEGA) gel
  • the kit can be used to purify DNA fragments with a size of 400 bp according to the instructions to obtain T
  • Nor1-F 5'-ACCGCTACGCCGGCACTCTCGGCAC-3';
  • Nor1-R 5'-GTTGGCCGCCAGCTTCGACACTCCG-3'.
  • the upstream and downstream primers (Ph-F, Ph-R) and probes of aflatoxin ( Ph-probe) sequence Tq-nor-1 upstream and downstream primers (Tq-nor1-F, Tq-nor1-R) and probe (Tq-probe) sequence, then according to the primers and probes in the double RT-PCR reaction.
  • the design principles and precautions were verified with Oligo7.0 primer analysis software.
  • TM values of all primers should be set to the same or close level, and the TM values of all probes should be as close as possible and greater than the primers TM value by about 5 ⁇ 10 °C; due to the synchronization of the same system Reaction to ensure that all primers and probes are not easy to form dimers; BLAST search to ensure that the primers and probes are specific to the target.
  • RT-PCR amplification of two single-layer aflatoxin anti-idiotype Nanobody phage-specific DNA fragments and nor-1 gene DNA fragments the reaction components are directly mixed without adding other components, and double RT-
  • Figure 1A The results of the PCR reaction are shown in Figure 1A. From the figure, we can see that VHH 2-5 phage DNA molecule amplification is significantly inhibited.
  • the amplification efficiency and target sequence of different amplicons may be different.
  • the amplification of samples with low amplification efficiency or low-abundance target sequences may be amplified by high-amplification samples or higher-abundance targets. Sequence suppression.
  • the present invention optimizes the RT-PCR reaction conditions of the method for the simultaneous detection of aflatoxin production and Nor-1 gene transcription.
  • concentrations of DNA polymerase, MgCl 2 and dNTPs are optimized.
  • the results shown in Figure 1B show that at a VHH 2-5 phage concentration of 10 6 pfu/mL, after additional addition of dNTPs and MgCl 2 , the cycle threshold Ct is advanced, and the amplification curve appears at an earlier cycle number. This indicates that the amplification of VHH 2-5 bacteriophage DNA molecules is improved.
  • Figure 1C shows that when the DNA polymerase is increased from 0.25U to 1.0U, the efficiency of phage amplification is also significantly improved. Therefore, according to the principle that the threshold cycle Ct appears earlier and the amplification curve is closer to the S-type in the exponential period, the preferred dosage range of the DNA polymerase, MgCl 2 and dNTPs of the present invention is:
  • DNA polymerase 0.5U ⁇ 1.0U
  • MgCl 2 1mM ⁇ 2mM
  • dNTPs 200uM ⁇ 400uM.
  • the present invention finally provides optimized amplification reaction parameters, as shown in Table 3 for details.
  • the amplification efficiency of the nor-1 gene is 90.25% similarly.
  • the amplification efficiency E meets the requirements of the range of 90%-105%, and the correlation coefficient of the amplification efficiency standard curve R 2 >0.99. Therefore, the optimized simultaneous RT-PCR system can be used for the simultaneous and efficient amplification of VHH2-5 bacteriophage and nor-1 gene.
  • the commercially-available aflatoxin monoclonal antibody 1C11 (hybridoma cell line 1C11 with the deposit number CCTCC NO: C201013 is secreted and produced by PBS, and the patent application number CN201010245095.5 is specifically reported)) is diluted to 1.0 ⁇ g/mL, add it to 96-well microplate with a micropipette, 100 ⁇ L per well, incubate at 4°C overnight ( ⁇ 12h), wash the plate 3 times with PBST; block: block with blocking solution, 300 ⁇ L per well, Incubate at 37°C for 45min, wash the plate 3 times with PBST;
  • Blocking block with blocking solution, 300 ⁇ L per well, incubate at 37°C for 45min, wash the plate 3 times with PBST;
  • the fluorescence quantitative system software will give different eluents corresponding to different amounts of aflatoxin.
  • OriginPro8.0 software The four-parameter logistic regression is used as the S-type standard curve for aflatoxin quantification, as shown in FIG. 3A.
  • the Aspergillus flavus strain N73 deposited by the research center was used as a highly virulent strain, which was used in the preparation of Tq-nor1 in this study.
  • Other Aspergillus flavus strains can be used to amplify the DNA fragment Tq-nor1 with a size of 400 bp using the "obtaining method of the nor-1 gene DNA fragment Tq-nor1" described above, which can be used to establish the quantification of nor-1 gene transcription Alternative strains for the standard curve.
  • Tq-nor1 After detecting the concentration of Tq-nor1 with a spectrophotometer (NanoDrop 2000, Thermo Scientific, USA), the copy number of Tq-nor1 was calculated. Tq-nor1 as a known amount of samples was serially diluted (10 2 ⁇ 10 8 copies), then performed RT-PCR amplification, using Origin Pro 8.0 software to take the logarithm of the Tq-nor1 standard series copy number as the abscissa, Ct The value is the ordinate, and regression analysis is performed, as shown in Figure 3B is the quantitative standard curve of nor-1 gene transcription.
  • FIG. 3A The quantitative standard curve of aflatoxin is shown in Figure 3A. It can be seen from the figure that the detection limit of simultaneous RT-PCR quantitative aflatoxin B1 LOD (indicated by IC 10 ) is 0.018ng/mL, so the established RT-PCR quantitative detection of aflatoxin B1 has high sensitivity.
  • Figure 3B reveals that synchronous RT-PCR can quantify the nor-1 gene copy number range from 10 2 to 10 8 , which fully confirms that the established synchronous RT-PCR has obvious advantages in the low level of absolute quantification of nor-1 gene. .
  • the cross-reaction rates against aflatoxins B1, B2, G1, and G2 are 100%, 101%, 34%, and 12%, respectively. This shows that this method can achieve the determination of the total amount of aflatoxin. Among them, the cross-reaction rates of aflatoxins G1 and G2 are 34% and 12%, respectively, but this does not affect the application of this method in identifying the virulence of aflatoxin, because the aflatoxin strain only produces group B aflatoxins.
  • the cross-reaction rate of aflatoxin B2 is 101%, and the established RT-PCR method for quantifying aflatoxin B2 has higher sensitivity, which to a certain extent improves the reliability of the established RT-PCR method for identifying the virulence of Aspergillus flavus Sex, because aflatoxin strains can produce both aflatoxin B1 and aflatoxin B2. Therefore, using established RT-PCR to identify the virulence of Aspergillus flavus strains is an assessment of the overall yield of B1 and B2.
  • Aflatoxin B1 standard and nor-1 gene DNA fragment Tq-nor-1 were added simultaneously in 2mL blank PDB medium. Mix by shaking. After mixing, place the mixture at 4°C in the dark for 4 days. After 4 days, the mixed solution was diluted 20 times, and the content of aflatoxin B1 and Tq-nor-1 in the mixed solution was simultaneously detected by RT-PCR. Set 3 repetitions in the group on the same day, and set 3 repetitions in the group on different days. See Table 3 for the added recovery results:
  • RNA extraction was performed according to the instructions of the RNA extraction kit (RNeasy Plant Mini Kit). Then use QuantiTect reverse transcription kit to synthesize cDNA.
  • the cDNA solution was diluted 100-1000 times to replace Tq-nor1 in the synchronous RT-PCR amplification system, and used as one of the amplification templates to perform synchronous RT-PCR amplification to determine the transcription amount of nor-1 gene. After 10 times (w/v) BSA/PBS dilution of the strain culture solution, the aflatoxin standard in the immune reaction was used to participate in the immune competition reaction.
  • the phagemid in the eluate after the competition reaction was used as synchronous RT-PCR
  • Another template of the amplification system was subjected to simultaneous RT-PCR amplification to determine the strain's toxin production.
  • the synchronous RT-PCR method was used to quantify the production of 17 strains of Aspergillus flavus and the expression level of nor-1 gene. The results are shown in Table 4.
  • the comparison results of different detection methods indicate that the established quantitative results of synchronous RT-PCR are reliable, and can be used for the simultaneous analysis of the toxin production of Aspergillus flavus strain and the transcription of nor-1 gene.
  • the ratio of aflatoxin production to nor-1 gene expression level is more reliable for evaluating the virulence of aflatoxin.
  • AFT/Nor-1 The toxin production volume of the strain was taken as the ordinate, and the logarithmic value of nor-1 gene expression and the AFT/Nor-1 ratio were plotted as the abscissa, respectively.
  • the correlation between aflatoxin production and Nor-1 gene expression is shown in Figure 6-A.
  • the toxin production is >150ng/mL; intermediate toxin production: 50 ⁇ toxicity production ⁇ 150ng/mL; low-production production: toxicity production ⁇ 50ng/mL; non-toxicity production: 0; Toxicity appraisal scope:
  • Toxigenic strains 6.5 ⁇ AFT/Nor-1 ⁇ 16.4;
  • AFT/Nor-1 the ratio of the amount of toxin produced to the amount of nor-1 transcript
  • AFT the first batch of aflatoxin production
  • Nor-1 the second batch of Nor-1 gene record
  • Ratio the third batch of AFT/Nor-1 ratio
  • the aflatoxin production and Nor-1 gene record in the table are the average of 5 repeated experiments in the group.
  • the method for identifying and evaluating the virulence of aflatoxin-producing strains proposed in the present invention namely AFT/Nor-1 ratio identification, is a more accurate and reliable method for identifying and evaluating the virulence of Aflatoxin strains .

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Abstract

一种用来鉴定评价黄曲霉毒素产毒菌株产毒力的方法，确定了黄曲霉毒素产量与Nor-1基因转录量比值具有非常好的相对稳定性，建立了黄曲霉菌株产毒力鉴定模型，获得了黄曲霉菌产毒力与黄曲霉毒素产量和Nor-1基因转录量比值AFT/Nor-1之间的回归方程，通过确定AFT/Nor-1，可以实现对黄曲霉菌株产毒力的快速、准确的鉴定评价，对黄曲霉毒素污染预警与防控具有重要意义；同时建立了黄曲霉毒素产量和Nor-1基因转录量同步检测RT-PCR方法，基于同步检测RT-PCR方法获得的AFT/Nor-1结果可靠、准确，可以作为判定黄曲霉菌株产毒力的鉴定指标。

Description

一种用来鉴定评价黄曲霉毒素产毒菌株产毒力的方法 技术领域
本发明属于生物领域，具体涉及一种用来鉴定评价黄曲霉毒素产毒菌株产毒力的方法。
背景技术
黄曲霉毒素是迄今发现毒性最强的一类真菌毒素，以黄曲霉毒素B1为例，其毒性是氰化钾的10倍，砒霜的68倍，被国际癌症组织列为I类致癌物。黄曲霉毒素极易污染花生、玉米、稻米等粮油作物产品，还污染核桃、开心果以及中药材等众多植物产品。国内外发生过多起黄曲霉毒素引发的人畜群体中毒事件。据国际癌症研究署(IARC)最新报道，全球仅发展中国家就有约5亿人口饱受黄曲霉毒素暴露风险。我国是黄曲霉毒素污染较重地区。农业部多年普查结果表明，我国主要农作物产品受黄曲霉毒素污染呈持续加重趋势，严重地区毒素含量超过限量标准的数百倍；虽然强产毒菌株占比不到20％，但却已成为威胁农作物产品质量安全的重大隐患。
黄曲霉毒素主要由黄曲霉、寄生曲霉等真菌产生。已有研究表明，不同黄曲霉菌株产生黄曲霉毒素的能力——即产毒力可相差数百倍，强产毒菌株是造成高污染的重要源头，但至今缺乏特异性快速鉴别强产毒菌株的有效方法。目前鉴别黄曲霉菌株产毒力的方法主要有两类：一类是培养菌株一定时间后直接通过测定其产生黄曲霉毒素的量来评价其产毒能力。这类方法一是时间长，必须首先分离出菌株，再培养，最后通过检测黄曲霉毒素含量进行评价；二是黄曲霉毒素的生物合成受影响因素非常多且复杂，同一菌株培养不同培养批次之间的黄曲霉毒素产量差异大，结果难以用作表征菌株本应恒定的自身产毒特性，从而影响该方法鉴别评价结果的准确可靠性。另一类方法是通过测定产毒相关基因转录水平来评价菌株产毒力，有文献报道检测Nor-1基因评价产毒力。但是，这类方法的不足之处在于：在自然状态下，就有Nor-1基因以外的产毒相关基因缺失，从而导致即使检测到Nor-1基因表达，仍然是天然不产黄曲霉毒素，从而导致这类方法的假性结果。
综上所述，客观准确地鉴定评价黄曲霉毒素产毒菌株产毒力一直是就而未决的世界性难题。
发明内容
本发明针对现有技术的不足，目的在于提供一种用来鉴定评价黄曲霉毒素产毒菌株产毒力的方法。
为实现上述发明目的，本发明采用的技术方案为：
一种用来鉴定评价黄曲霉毒素产毒菌株产毒力的方法，测定黄曲霉毒素产量与Nor-1基因转录量，获得黄曲霉毒素产量与Nor-1基因转录量比值，根据黄曲霉毒素产量与Nor-1基因转录量比值鉴定评价黄曲霉毒素产毒菌株产毒能力。
按上述方案，所述黄曲霉毒素产量的测定方法：培养黄曲霉菌株，取黄曲霉菌的孢子进行振荡培养，培养结束后，过滤取滤液，测定滤液中黄曲霉毒素浓度。
按上述方案，所述培养黄曲霉菌株所用的培养基为CDA培养基，培养条件：28℃、90％湿度下培养10天；
所述取黄曲霉菌的孢子振荡培养所用的培养基为马铃薯葡萄糖液体培养基；培养条件：在28℃，200rpm条件下振荡培养96h；
按上述方案，采用免疫亲和净化-高效液相色谱标准方法，测定所述黄曲霉菌的孢子振荡培养后滤液中的黄曲霉毒素浓度。
按上述方案，所述Nor-1基因转录量的测定方法：培养黄曲霉菌株，取黄曲霉菌的孢子进行振荡培养，培养结束后，过滤取黄曲霉菌菌丝球，干燥后得到干菌体，采用常规的Nor-1基因转录量测定方法，测定干菌体中Nor-1基因转录量。
按上述方案，所述黄曲霉毒素产量与Nor-1基因转录量还可以通过同步检测RT-PCR方法获得，具体的步骤包括：
(1)黄曲霉毒素定量S型标准曲线的建立：免疫反应阶段，在黄曲霉毒素单克隆抗体1C11包被量一定的情况下，采用不同浓度的黄曲霉毒素标品与V2-5噬菌体竞争结合1C11，免疫反应结束后，将结合在1C11上的噬菌体洗脱，不同浓度的黄曲霉毒素标品对应洗脱不同量的噬菌体，洗脱液中的噬菌体在PCR加热的过程中会释放DNA分子，释放的DNA分子作为RT-PCR反应中的扩增靶标，采用所述试剂盒将各洗脱液分别进行RT-PCR扩增反应，扩增反应结束后获得不同的Ct值，以黄曲霉毒素浓度的对数值为横坐标，以Ct值为纵坐标，进行回归分析，得到黄曲霉毒素的定量S型标准曲线；
(2)Nor-1基因转录量RT-PCR标准曲线的建立：将nor-1基因DNA片段Tq-nor1已知拷贝数的样本连续稀释成不同拷贝数，然后采用所述试剂盒对各拷贝数Tq-nor1分别进行RT-PCR扩增反应，扩增反应结束后获得不同的Ct值，以Tq-nor1拷贝数的对数值为横坐标，以Ct值为纵坐标，进行回归分析，得到nor-1基因转录量的定量标准曲线；
(3)培养黄曲霉菌株，取黄曲霉菌孢子进行振荡培养，培养结束后，过滤取菌株培养液和黄曲霉菌丝球；将菌株培养液稀释一定倍数后，替代步骤(1)所述免疫反应 中的黄曲霉毒素标品参与免疫竞争反应，竞争反应后将结合在1C11上的噬菌体洗脱，将洗脱液中的V2-5噬菌体作为同步RT-PCR扩增反应中定量黄曲霉毒素的扩增模板；另将黄曲霉菌丝球干燥后，提取总RNA并反转录成cDNA，将该cDNA稀释一定倍数后作为同步RT-PCR扩增反应中扩增Nor-1基因的模板；
(4)以所述V2-5噬菌体和所述cDNA为模板进行RT-PCR同步扩增反应，扩增反应结束后获得两个Ct值，将两个Ct值分别代入黄曲霉毒素的定量S型标准曲线和nor-1基因转录量的定量标准曲线，换算得到黄曲霉毒素浓度和nor-1基因转录量，以此确定黄曲霉毒素产量与Nor-1基因转录量的比值。
按上述方案，所述表面展示黄曲霉毒素抗独特型纳米抗体的噬菌体为噬菌体VHH2-5，所述黄曲霉毒素单克隆抗体为黄曲霉毒素单克隆抗体1C11。
按上述方案，所述同步RT-PCR扩增反应的反应体系中，上下游引物：Ph-F、Ph-R、Tq-nor1-F、Tq-nor1-R的终浓度均为300～400nM；荧光探针：Ph-probe和Tq-probe的终浓度200～400nM；DNA聚合酶最终用量：0.5U～1.0U；MgCl 2终浓度：1mM～2mM；dNTPs终浓度：200uM～400uM。
按上述方案，所述上游引物Ph-F，核苷酸序列如SEQ ID NO.1所示；下游引物Ph-R，核苷酸序列如SEQ ID NO.2所示；荧光探针Ph-probe，核苷酸序列如SEQ ID NO.3所示；所述上游引物Tq-nor1-F，核苷酸序列如SEQ ID NO.4所示；下游引物Tq-nor1-R，核苷酸序列如SEQ ID NO.5所示；荧光探针Tq-probe，核苷酸序列如SEQ ID NO.6所示。
按上述方案，所述同步RT-PCR扩增反应的反应体系包括：通用型探针qPCR预混液5μL、Ph-F 0.1μL、Ph-R 0.1μL、Ph-probe 0.1μL、噬菌体模板2μL、Tq-nor1-F 0.1μL、Tq-nor1-R 0.1μL、Tq-probe 0.1μL、Nor-1基因模板1μL、DNA聚合酶0.2μL、MgCl 2 0.8μL、dNTPs 0.2μL，加H 2O补齐10μL。
按上述方案，所述RT-PCR同步扩增反应的条件为95℃、5min；95℃、10s，60℃30s，40个循环。
按上述方案，所述黄曲霉毒素的定量S型标准曲线中黄曲霉毒素浓度范围为33.33ng/mL～1.69pg/mL，黄曲霉毒素的最低检测线LOD为0.018ng/mL；所述nor-1基因转录量的定量标准曲线中nor-1基因拷贝数范围为10 2～10 8。
按上述方案，将所述黄曲霉毒素产毒菌株产毒力定义为Y，将黄曲霉毒素产量与Nor-1基因转录量(AFT/Nor-1)的比值定义为X，黄曲霉菌株产毒力鉴定方程为：Y＝10.14X-16.20，根据高产毒菌株，产毒量>150ng/mL；中产毒：50<产毒量<150 ng/mL；低产毒：产毒量<50ng/mL；不产毒：0；代入方程计算得到产毒力鉴定范围：AFT/Nor-1>16.4，为高产毒菌株；6.5<AFT/Nor-1<16.4，为中产毒菌株；0<AFT/Nor-1<6.5，为低产毒菌株；AFT/Nor-1＝0，为不产毒菌株。
本发明的有益效果如下：
(1)本发明研究探明了黄曲霉毒素产量与Nor-1基因转录量比值具有非常好的相对稳定性，建立了黄曲霉菌株产毒力鉴定模型，即获得了黄曲霉菌产毒力与黄曲霉毒素产量和nor-1基因转录量比值(AFT/Nor-1)之间的回归方程，通过确定AFT/Nor-1比值，可以实现对黄曲霉菌株产毒力的快速、准确的鉴定评价，对黄曲霉毒素污染预警与防控具有重要意义；
(2)本发明建立了同步检测黄曲霉菌株产毒量和nor-1基因转录量的同步RT-PCR检测方法，利用本发明所述同步检测RT-PCR方法所得黄曲霉菌株产毒量、nor-1基因转录量与采用HPLC定量黄曲霉毒素、Nanodrop定量Nor-1基因转录量的结果存在良好的线性关系，因此，基于同步检测RT-PCR方法获得的AFT/Nor-1(黄曲霉菌株产毒量/nor-1基因转录量)的比值结果可靠、准确，可以作为判定黄曲霉菌株产毒力高低的鉴定指标；
(3)本发明所述黄曲霉菌株产毒量和nor-1基因转录量的同步检测RT-PCR方法中，试剂用量更少，成本更低，可以实现高通量检测；所述同步检测RT-PCR方法精简了分析模式，优化了实验过程和结构，并为黄曲霉毒素及其合成通路中其他小分子物质的同步分析提供了检测平台和理论基础。
附图说明
图1为优化同步RT-PCR反应中DNA聚合酶、dNTPs和MgCl 2浓度。
图2为同步RT-PCR扩增效率评估。
图3为同步RT-PCR定量黄曲霉毒素B1和nor-1基因转录量的定量标准曲线。
图4为同步RT-PCR定量：与黄曲霉毒素B1、B2、G1、G2、ZEN、DON、FB1交叉反应率。
图5为同步RT-PCR与HPLC和Nanodrop定量结果比对。
图6为黄曲霉毒素产量与Nor-1基因表达量相关关系(A)、黄曲霉毒素产量与AFT/Nor-1比值相关关系(B)。
具体实施方式
为了更好地理解本发明，下面结合实施例进一步阐明本发明的内容，但本发明的内容不仅仅局限于下面的实施例。
实施例1
一、研究探明了黄曲霉毒素产量与Nor-1基因转录量比值具有非常好的相对稳定性
称取1g NaNO 3、1g K 2HPO 4、0.5g MgSO 4·7H 2O、0.5g KCl、0.01g FeSO 4、30g葡萄糖和20g琼脂粉，用去离子水定容至总体积为1000ml，121℃高温蒸汽灭菌30min，制备成CDA培养基。用CDA固体培养基于28℃、90％湿度下培养黄曲霉菌株10天，然后用20％吐温-20洗涤培养平板以获得黄曲霉菌孢子溶液。采用血球计数板计数法，将黄曲霉菌孢子溶液用旋涡震荡仪震荡均匀，用显微镜对黄曲霉菌孢子溶液显微计数。
将15mL马铃薯葡萄糖液体培养基置于50mL三角瓶，121℃高压灭菌30min，根据计数结果，加入黄曲霉菌孢子溶液至终浓度为每毫升1×10 5个孢子，在28℃，200rpm条件下振荡培养96h。用双层滤纸过滤培养液，过滤得到滤液(保存备用)和黄曲霉菌菌丝球。针对黄曲霉菌菌丝球，用滤纸挤压掉多余水分，用烘箱在65℃条件下干燥12h，得到干菌体，冷却至室温后置于-70℃条件下保存备用，过滤得到的滤液4℃条件下保存备用。
采用免疫亲和净化-高效液相色谱标准方法，测定黄曲霉菌孢子振荡培养后得到的滤液(上述保存备用的)中黄曲霉毒素浓度，采用常规的Nor-1基因转录相对量测定方法，测定上述保存备用的干菌体中Nor-1基因转录相对量。
采用如上所述同样的操作，7个黄曲霉菌株在不同时间段先、后培养测定了两个批次结果，测定结果见表1。
表1黄曲霉菌株产黄曲霉毒素与Nor-1基因转录相对量测定结果
Figure PCTCN2019121785-appb-000001
根据表1测定结果，两个批次黄曲霉毒素浓度差异较大，因此单凭黄曲霉毒素产量不能满足评价黄曲霉菌株产毒力；有的黄曲霉菌株Nor-1基因转录相对量高时，竟然有产毒力反而相对低的情况；另外不产毒菌株也能转录Nor-1基因，因此单凭Nor-1基因也无法满足评价黄曲霉菌株产毒力。出乎意料的是，在表1中，当把黄曲霉毒素浓度值和Nor-1基因转录相对量值相除后得到的比值——即表1中的[AFT]/[nor-1]数值，发现呈现出了很强的规律性，不仅两次培养测定批次得到的7个菌株产毒力顺序一致，而且每个菌株的[AFT]/[nor-1]数值具有相对稳定性，从而可满足黄曲霉菌株产毒力评价。
从表1数据中可以看出，如果采用黄曲霉毒素浓度值和Nor-1基因转录相对量值的比值来评价黄曲霉菌株产毒力，准确性和可靠性明显优于单独采用黄曲霉毒素浓度值或者单独采用和Nor-1基因转录相对量值法。
实施例2黄曲霉毒素产量与Nor-1基因转录量的同步检测RT-PCR方法的建立
利用已有的表面展示黄曲霉毒素抗独特型纳米抗体的噬菌体VHH 2-5和nor-1基因DNA片段Tq-nor1，建立了黄曲霉毒素产量和Nor-1基因转录量同步检测RT-PCR方法，根据上述检测结果作为鉴定评价黄曲霉菌株产毒力的科学依据，为快速鉴定评价黄曲霉菌株产毒力提供了方法支撑。这些具体步骤如下。
所述表面展示黄曲霉毒素抗独特型纳米抗体的噬菌体VHH 2-5为中国农业科学院油料作物研究所质量检测中心研制，已在期刊文献“Yanru Wang；Peiwu Li；Zuzana Majkova；Candace R.S.Bever；Hee Joo Kim；Qi Zhang；Julie E.Dechant；Shirley J.Gee；Bruce D.Hammock；Isolation of Alpaca Anti-Idiotypic Heavy-Chain Single-Domain Antibody for the Aflatoxin Immunoassay；Analytical Chemistry,2013,8298-8303”报道过，本实施例所使用的噬菌体是实验室事先保存在大肠杆菌ER2738中，通过扩增获得。扩增方法如下：
从保有纳米抗体噬菌体VHH 2-5的ER2738单菌落平板上随机挑取单克隆，接种到含有1mL SB-氨苄液体培养基中，于37℃恒温摇床中225rpm培养过夜；将上述过夜培养菌液加入到100mL SB-氨苄液体培养基中，225rpm，37℃培养至OD 600＝0.5-0.6；加入1.5ml M13KO7辅助噬菌体(滴度在1×10 11-1×10 12pfu/mL)到培养好的菌液中，37℃静置30min；在菌液中加入终浓度为70μg/mL的卡纳霉素，37℃，225rpm，振荡培养过夜；4℃，10000rpm离心过夜培养的菌液15min，取上清，并转移至干净的离心瓶中；加入1/4体积的PEG/NaCl，于冰上静置2h；4℃，10000rpm离心30min，将沉淀用2mL 0.5％BSA/PBS重悬，然后于12000rpm离心5min，取上清，将上清用 0.22μm的滤器过滤并与等体积灭菌甘油混合，分装，测滴度。滴度测定方法如下：
从ER2738单菌落平板上随机挑取单克隆，接种到含有0.04mg/mL四环素的LB液体培养基中，于37℃恒温摇床中225rpm培养过夜；取上述过夜培养菌液20μL加至2mL SB培养基，225rpm，37℃培养至OD 600≈1；用LB液体培养基对需要测定滴度的噬菌体进行梯度稀释；每个稀释度分别取10μL，加入到100μL OD 600≈1的ER2738菌液中，37℃静置20min，使噬菌体侵染大肠杆菌；将侵染后的大肠杆菌菌液涂布于LB-氨苄平板上，37℃恒温培养箱中正置30min，然后倒置培养过夜；选取单菌落数量在100个左右的进行平板计数，按照以下公式来计算噬菌体的滴度：
Figure PCTCN2019121785-appb-000002
所述nor-1基因DNA片段Tq-nor1的获得方法：将15mL马铃薯葡萄糖液体培养基置于50mL三角瓶，121℃高压灭菌30min，加入N73号黄曲霉菌的孢子液至终浓度为1×10 5ml -1，在28℃，200rpm条件下振荡培养96h后；双层滤纸挤压掉菌丝球培养液，65℃干燥12h，液氮速冻，研磨成粉；准确称量200mg菌丝粉末，用DNA提取试剂盒(DNeasy Plant Mini Kit)，按照试剂盒说明书提取N73号菌株的基因组DNA，用如下引物扩增出大小为400bp的nor-1基因片段产物，并用E.Z.N.A.TM(OMEGA)凝胶提取试剂盒，按照说明书纯化大小为400bp的DNA片段，即可获得Tq-nor1。
Nor1-F:5'-ACCGCTACGCCGGCACTCTCGGCAC-3'；
Nor1-R:5'-GTTGGCCGCCAGCTTCGACACTCCG-3'。
一、黄曲霉毒素产量与Nor-1基因转录量同步检测RT-PCR方法建立
1、黄曲霉毒素产量与Nor-1基因转录量同步检测RT-PCR的引物和探针序列设计以表面展示黄曲霉毒素抗独特型纳米抗体的噬菌体VHH2-5释放的DNA片段和nor-1基因的DNA片段Tq-nor1为模板进行RT-PCR同步扩增，通过同步RT-PCR的扩增结果确定黄曲霉毒素产量与Nor-1基因转录量的比值。
根据表面展示黄曲霉毒素抗独特型纳米抗体的噬菌体VHH 2-5中编码纳米抗体的序列和nor-1基因序列，黄曲霉毒素的上下游引物(Ph-F,Ph-R)和探针(Ph-probe)序列、Tq-nor-1的上下游引物(Tq-nor1-F,Tq-nor1-R)和探针(Tq-probe)序列，然后根据双重RT-PCR反应中引物及探针设计原则和注意事项，用Oligo7.0引物分析软件进行验证。
引物及探针验证原则：所有引物的TM值应设定相同或接近的水平，且应尽可能使所有探针的TM值接近并大于引物TM值约5～10℃；由于是同一系统的同步反应，确保所有引物和探针不易形成二聚体；BLAST搜索，确保引物和探针对目的靶点具有特异性。
下述引物及探针序列确定满足以上原则要求，如表2总结所示。
表2双重荧光定量RT-PCR引物和探针序列
Figure PCTCN2019121785-appb-000003
2、黄曲霉毒素产量与Nor-1基因转录量同步RT-PCR检测方法的反应参数
同步RT-PCR反应参数不同于单一基因扩增的RT-PCR，因为要充分考虑反应体系之间相互干扰。首先，将两种单重——黄曲霉毒素抗独特型纳米抗体噬菌体特异DNA片段和nor-1基因DNA片段的RT-PCR扩增，反应组分直接混合不添加其他组分，进行双重RT-PCR反应，结果如图1A所示，从图中我们看到VHH 2-5噬菌体DNA分子扩增受到明显的抑制。双重RT-PCR反应中，不同扩增物的扩增效率以及靶序列都可能不同，扩增效率低的样品或低丰度靶序列的扩增可能被高扩增样品或更高丰度的靶序列抑制。
为此，本发明对黄曲霉毒素产量与Nor-1基因转录量同步检测方法的RT-PCR反应条件进行优化。本发明中优化了DNA聚合酶、MgCl 2和dNTPs的浓度。图1B所示的结果表明，VHH 2-5噬菌体浓度为10 6pfu/mL时，额外添加dNTPs和MgCl 2后，循环阈值Ct前移，在较早循环数时出现扩增曲线，由此，说明提高了VHH 2-5噬菌体DNA分子的扩增。图1C显示当DNA聚合酶从0.25U增加到1.0U时，噬菌体的扩增效率也显著提高。因此，依据阈值循环Ct越较早出现和扩增曲线在指数期越接近S型的原则，本发明DNA聚 合酶、MgCl 2和dNTPs的优选用量范围为：
DNA聚合酶：0.5U～1.0U；MgCl 2：1mM～2mM；dNTPs：200uM～400uM。
本发明根据上述研究结果，最终给出了优化的扩增反应参数，具体见表3。
表3黄曲霉毒素与Nor-1基因转录量同步检测方法的RT-PCR反应参数
Figure PCTCN2019121785-appb-000004
3、黄曲霉毒素产量与Nor-1基因转录量同步检测RT-PCR方法的扩增效率
系列稀释的噬菌体和系列稀释的nor-1的同步RT-PCR扩增结果见同步RT-PCR扩增曲线(RFU，相对荧光单位)，如图2A所示。图2B为根据扩增曲线得到的扩增效率标准曲线，噬菌体VHH 2-5扩增效率的标准曲线斜率-3.37，根据扩增效率E的计算公式(E＝[10 1/-slope-1]×100％)计算，当扩增效率E为98.03％时，VHH 2-5噬菌体的可检测的浓度范围是10 9～10 3pfu/mL。当nor-1基因DNA片段Tq-nor1的拷贝数在10 2-10 8拷贝时，同理得nor-1基因扩增效率为90.25％。扩增效率E均满足90％-105％的范围要求，且扩增效率标准曲线相关系数R 2>0.99。因此，优化的同步RT-PCR体系可用于VHH2-5噬菌体和nor-1基因的同步高效扩增。
二、定量标准曲线建立及同步RT-PCR方法评估
1.黄曲霉毒素定量的S型标准曲线
1.1免疫反应
(1)包被：用PBS将市售黄曲霉毒素单克隆抗体1C11(保藏编号为CCTCC NO：C201013的杂交瘤细胞株1C11分泌产生，专利申请号为CN201010245095.5有具体报道))稀释至1.0μg/mL，用微量移液器加入到96-well酶标板中，每孔100μL，于4℃孵育过夜(≈12h)，PBST洗板3次；封闭：用封闭液封闭，每孔300μL，于37℃孵育45min，PBST洗板3次；
(2)封闭：用封闭液封闭，每孔300μL，于37℃孵育45min，PBST洗板3次；
(3)竞争：将黄曲霉毒素B1标品，用100％纯甲醇稀释至浓度为200ng/mL，然后用10％(v/v)甲醇/PBS 3倍梯度倍比稀释黄曲霉毒素B1标准溶液，使浓度范围为33.33ng/mL-1.69pg/mL；然后将50μL已知浓度的VHH 2-5噬菌体(1.0×10 10cfu/mL)与50μL系列浓度的黄曲霉毒素B1混合，将100μL混合物加入到96-well酶标版微孔，在37℃温育1h后，用PBST洗涤酶标板10次；
(4)洗脱：每孔注入90μL噬菌体洗脱液，37℃静置温浴15min，用微量移液器轻轻吹打并转移出含有噬菌粒的洗脱液；
(5)中和：90μL移出液与10μL中和液混匀使混合液成中性——加入中和液的体积根据洗脱液和中和液的实际pH值调整，保证混合液成中性；
1.2建立标准曲线：免疫反应阶段，黄曲霉毒素单克隆抗体1C11包被量一定的情况下，不同浓度的黄曲霉毒素会与不同量的VHH 2-5噬菌体竞争结合1C11，黄曲霉毒素浓度越大，噬菌体结合1C11的机会就越小，结合量越低；免疫反应结束后，将结合在1C11上的噬菌体洗脱，洗脱液中的噬菌体数量和黄曲霉毒素浓度相关，洗脱液中的噬菌粒在PCR反应加热的过程会释放DNA分子，释放的DNA分子作为RT-PCR反应中的扩增靶标，扩增反应后荧光定量系统软件会给出不同量黄曲霉毒素对应的不同洗脱液中不同数量的噬菌体扩增的Ct值，系列稀释的不同浓度的黄曲霉毒素(33.33ng/mL～1.69pg/mL)得到的Ct值对黄曲霉毒素浓度的对数值，用Origin Pro 8.0软件进行四参数逻辑回归，作为黄曲霉毒素定量的S型标准曲线，如图3A所示。
2.nor-1基因转录量的定量标准曲线建立
取黄曲霉菌株，本实施例采用本研究中心保藏的N73号黄曲霉菌株，为高产毒菌株，用于本研究中Tq-nor1制备。其他黄曲霉菌株，能用上文叙述的“nor-1基因DNA片段Tq-nor1的获得方法”扩增出大小为400bp的DNA片段Tq-nor1，均可作为建立nor-1基因转录量的定量标准曲线的备选菌株。分光光度计(NanoDrop 2000,Thermo Scientific,U.S.A.)检测Tq-nor1浓度后，计算得到Tq-nor1的拷贝数。Tq-nor1作为已知量样本进行连续稀释(10 2～10 8copies)后，进行RT-PCR扩增，用Origin Pro 8.0软件以Tq-nor1标准样系列拷贝数的对数值为横坐标，Ct值为纵坐标，进行回归分析，如图3B为nor-1基因转录量的定量标准曲线。
黄曲霉毒素的定量标准曲线如图3A所示。由图可知，同步RT-PCR定量黄曲霉毒素B1的检测限LOD(用IC 10表示)为0.018ng/mL，因此建立的RT-PCR定量检测黄曲霉毒素B1具有很高的灵敏度。此外，图3B揭示，同步RT-PCR可以定量nor-1基因拷贝数范围为10 2～10 8，这充分证实建立的同步RT-PCR在低水平的nor-1基因绝对定量方面具有明显的优势。
3.同步RT-PCR检测黄曲霉毒素的交叉反应率测定
黄曲霉毒素B2、G1、G2，玉米赤霉烯酮(ZEN)，脱氧雪腐镰刀菌烯醇(DON)，伏马毒素(FB1)标准品稀释为一系列浓度，与噬菌体VHH 2-5发生竞争免疫反应后，将酶标板孔底结合在单克隆抗体1C11上的噬菌体洗脱出来与Tq-nor1进行RT-PCR同步扩增，扩增得到的Ct值对应毒素不同浓度对数值做标准曲线，计算IC 50值，根据交叉反应计算公式％CR＝(IC 50AFB1/IC 50analyte)×100计算交叉反应率。如图4所示，针对黄曲霉毒素B1、B2、G1、G2的交叉反应率分别为100％、101％、34％和12％。由此可见，该方法可实现对黄曲霉毒素总量的测定。其中，黄曲霉毒素G1和G2的交叉反应率分别为34％和12％，但是不影响该方法在鉴定黄曲霉产毒力方面的应用，因为黄曲霉菌株仅产生B族类黄曲霉毒素。黄曲霉毒素B2的交叉反应率为101％，建立的RT-PCR方法定量黄曲霉毒素B2具有更高的灵敏度，这在一定程度上提高了建立的RT-PCR方法鉴定黄曲霉产毒力的可靠性，因为黄曲霉菌株即可产生黄曲霉毒素B1也可产生黄曲霉毒素B2。因而，用建立的RT-PCR鉴定黄曲霉菌株的产毒力，是对B1和B2综合产量的评定。
4.同步RT-PCR添加回收验证
在2mL空白PDB培养基同时添加黄曲霉毒素B1标品和nor-1基因DNA片段Tq-nor-1。震荡混匀，混合均匀后将混合液置于4℃避光放置4天。4天后，将混合液20倍稀释，用RT-PCR同步检测混合液中的黄曲霉毒素B1和Tq-nor-1的含量。同一天 组内设置3个重复，不同日期组间设置3个重复。添加回收结果见表3：
表3添加回收率测定
Figure PCTCN2019121785-appb-000005
黄曲霉毒素B1的添加回收率为88.37％～103.10％，Nor-1基因DNA片段Tq-nor-1的添加回收率为86.18％～98.17％，此结果表明，建立的同步RT-PCR在实际样品检测分析中，具有可靠的重复和再现性。
5.应用同步RT-PCR方法定量黄曲霉毒素菌株的产毒量和nor-1基因表达水平
本研究选取了17株具有不同产毒力的黄曲霉菌株。将15mL马铃薯葡萄糖液体培养基置于50mL三角瓶，121℃高压灭菌30min，加入黄曲霉孢子液至终浓度为1×10 5ml -1。在28℃，200rpm条件下振荡培养96h后，用双层滤纸过滤培养液，过滤得到菌株培养液和黄曲霉菌丝球，黄曲霉菌丝球用滤纸挤压掉多余水分，用烘箱在65℃条件下干燥12h，冷却至室温后，用液氮研磨成粉末，每个样本准确称量0.20mg菌丝粉末。根据RNA提取试剂盒(RNeasy Plant Mini Kit)说明书进行总RNA提取。然后用QuantiTect反转录试剂盒合成cDNA。该cDNA溶液100～1000倍稀释后代替同步RT-PCR扩增体系中的Tq-nor1，作为扩增模板之一进行同步RT-PCR扩增，测定nor-1基因转录量。菌株培养液用10％(w/v)BSA/PBS稀释10倍后，代替免疫反应中的黄曲霉毒素标品参与免疫竞争反应，竞争反应后洗脱液中的噬菌粒作为同步RT-PCR扩增体系的另一模板，进行同步RT-PCR扩增测定菌株产毒量。用同步RT-PCR方法定量17株黄曲霉菌的产毒量和nor-1基因表达水平，结果如表4。
表4同步RT-PCR定量17株黄曲霉菌的产毒量和nor-1基因表达水平结果
Figure PCTCN2019121785-appb-000006
为了验证结果的准确性，参考国家标准GB5009.22-2016方法，用HPLC法定量黄曲霉菌株产黄曲霉毒素量；同时，用分光光度计(Nanodrop)定量菌株nor-1基因表达量，定量结果如表4。并将结果与同步RT-PCR获得的结果进行比对，比对结果如图5。图5-A为RT-PCR与HPLC定量黄曲霉毒素比对结果，得到的线性回归方程为Y＝0.947X–3.84，线性回归分析产生了良好的相关性(R 2＝0.999)。图5-B为RT-PCR与Nanodrop定量Nor-1基因转录量比对结果，方法得到的线性回归方程为Y＝1.05X–1.18，相关系数R 2＝0.989。不同检测方法比对结果说明建立的同步RT-PCR定量结果可靠，可用于黄曲霉菌株产毒量和nor-1基因转录量同步分析。
实施例3
一、采用Nor-1转录量与AFT/Nor-1(产毒量与nor-1转录量比值)分别评价黄曲霉产毒力比较分析
通过结果比较分析我们发现，如果单从nor-1转录量来评价黄曲霉产毒力大小，鉴定结果不可靠。如黄曲霉菌株Pc124-2和Pc34-1，nor-1基因表达量拷贝数的对数值分别为7.85±0.52和6.75±0.58，两菌株nor-1基因表达水平相当；然而Pc124-2菌株的黄曲霉毒素产量为66.5±4.93ng/mL，Pc34-1产黄曲霉毒素的量仅为19.69±2.27ng/mL；此外，CY1，CY2，Pg28-1和Pc321-1-3菌株均未检测到黄曲霉毒素产生，然而，Pg28-1和Pc321-1-3表达的nor-1基因水平甚至比产黄曲霉毒素的一些菌株更高。
但是，黄曲霉毒素产生量和nor-1基因表达水平的比值用来评价黄曲霉菌产毒力的结果更可靠。为了进一步探明黄曲霉菌株的产毒力和nor-1基因表达水平，以及AFT/Nor-1之间的相关关系。分别以菌株产毒量为纵坐标，分别以nor-1基因表达量的对数值和AFT/Nor-1比值为横坐标作图。黄曲霉毒素产量与Nor-1基因表达量相关关系如图6-A所示，线性回归方程为：y＝36.37x–196.21，线性回归分析产生的相关性系数R 2＝0.693。然而，黄曲霉毒素产量与AFT/Nor-1比值相关关系如图6-B所示，线性回归方程为：y＝10.14x–16.20，线性回归分析产生的相关性系数R 2＝0.979。黄曲霉毒素产量与AFT/Nor-1比值产生非常好的相关性。
因此，本发明提出黄曲霉菌株产毒力鉴定方程为：y＝10.14x–16.20，其中X值代表AFT/Nor-1，Y为产毒力。
根据高产毒菌株，产毒量>150ng/mL；中产毒：50<产毒量<150ng/mL；低产毒：产毒量<50ng/mL；不产毒：0；然后根据回归方程计算得到产毒力鉴定范围：
高产毒菌株：AFT/Nor-1>16.4；
中产毒菌株：6.5<AFT/Nor-1<16.4；
低产毒菌株：0<AFT/Nor-1<6.5；
不产毒菌株：AFT/Nor-1＝0。
AFT/Nor-1(产毒量与nor-1转录量比值)鉴定产毒力结果稳定性验证
二、AFT/Nor-1(产毒量与nor-1转录量比值)鉴定产毒力结果稳定性验证
为了进一步验证AFT/Nor-1比值鉴定产毒力结果的稳定性，对5株黄曲霉菌株分别进行了黄曲霉毒素产生量和nor-1基因转录量的组内和组间实验分析。组内实验在同一天平行设置5个重复，5个重复平均值作为该批次菌株的产毒量和nor-1基因转录量。 组间实验设置3个不同日期，每个日期为一个批次，共设置3个批次。结果如下表5。
表5 AFT/Nor-1(产毒量与nor-1转录量比值)鉴定产毒力结果稳定性验证
Figure PCTCN2019121785-appb-000007
注： 1AFT:第一批次黄曲霉毒素产生量； 2Nor-1:第二批次Nor-1基因录量； 3Ratio:第三批次AFT/Nor-1比值；
*CV、 #CV、 &CV：分别为不同批次间检测黄曲霉毒素产生量、nor-1基因转录量和AFT/Nor-1比值变异系数；
@:变异系数(CV)值大于15％；
表内黄曲霉毒素产生量、Nor-1基因录量均为组内5个重复试验平均值。
从表中我们不难发现，不同批次培养检测的黄曲霉毒素的产生量和nor-1基因转录量差异较大。以菌株233-1为例，三个批次检测黄曲霉毒素的产量分别为10.07、19.69和25.32ng/mL，不同批次组间差异系数为42.00％。此外菌株IT-2、Pg56-1-2、N200和233-1产黄曲霉毒素的量，不同批次组间差异系数均大于17.00％。由此可见，单独从黄曲霉毒素的产生量评价菌株产毒力是不可靠的。同理分析，不同批次组间nor-1基因转录量发现，nor-1基因转录量差异也较大。同样证实仅仅从nor-1基因转录量鉴定评价菌株产毒力不可靠。然而，AFT/Nor-1比值的组间差异系数均小于13％，具有更好的稳定性。
综上说明，本发明中提出的用来鉴定评价黄曲霉毒素产毒菌株产毒力的方法，即AFT/Nor-1比值鉴定，是一更加准确可靠的鉴定评价黄曲霉菌株产毒力的方法。
显然，上述实施例仅仅是为清楚地说明所作的实例，而并非对实施方式的限制。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而因此所引申的显而易见的变化或变 动仍处于本发明创造的保护范围之内。

Claims (10)

  1. 一种用来鉴定评价黄曲霉毒素产毒菌株产毒力的方法，其特征在于，通过测定黄曲霉毒素产量与Nor-1基因转录量，获得黄曲霉毒素产量与Nor-1基因转录量比值，根据黄曲霉毒素产量与Nor-1基因转录量比值鉴定评价黄曲霉毒素产毒菌株产毒能力。
  2. 根据权利要求1所述的用来鉴定评价黄曲霉毒素产毒菌株产毒力的方法，其特征在于，所述黄曲霉毒素产量的测定方法：培养黄曲霉菌株，取黄曲霉菌的孢子进行振荡培养，培养结束后，过滤取滤液，测定滤液中黄曲霉毒素浓度。
  3. 根据权利要求1所述的用来鉴定评价黄曲霉毒素产毒菌株产毒力的方法，其特征在于，所述培养黄曲霉菌株所用的培养基为CDA培养基，培养条件：28℃、90％湿度下培养10天；所述取黄曲霉菌的孢子振荡培养所用的培养基为马铃薯葡萄糖液体培养基；培养条件：在28℃，200rpm条件下振荡培养96h。
  4. 根据权利要求1所述的用来鉴定评价黄曲霉毒素产毒菌株产毒力的方法，其特征在于，采用免疫亲和净化-高效液相色谱标准方法，测定所述黄曲霉菌的孢子振荡培养后滤液中的黄曲霉毒素浓度。
  5. 根据权利要求1所述的用来鉴定评价黄曲霉毒素产毒菌株产毒力的方法，其特征在于，所述Nor-1基因转录量的测定方法：培养黄曲霉菌株，取黄曲霉菌的孢子进行振荡培养，培养结束后，过滤取黄曲霉菌菌丝球，干燥后得到干菌体，采用常规的Nor-1基因转录量测定方法，测定干菌体中Nor-1基因转录量。
  6. 根据权利要求1所述的用来鉴定评价黄曲霉毒素产毒菌株产毒力的方法，其特征在于，所述黄曲霉毒素产量与Nor-1基因转录量还可以通过同步检测RT-PCR方法获得，具体方法包括如下步骤：
    (1)黄曲霉毒素定量S型标准曲线的建立：免疫竞争反应阶段，在黄曲霉毒素单克隆抗体包被量一定的情况下，采用不同浓度的黄曲霉毒素标品与表面展示黄曲霉毒素抗独特型纳米抗体的噬菌体竞争结合黄曲霉毒素单克隆抗体，免疫竞争反应结束后，将结合在黄曲霉毒素单克隆抗体上的表面展示黄曲霉毒素抗独特型纳米抗体的噬菌体洗脱，不同浓度的黄曲霉毒素标品对应洗脱不同量的表面展示黄曲霉毒素抗独特型纳米抗体的噬菌体，洗脱液中的噬菌体在PCR加热的过程中会释放DNA分子，释放的DNA分子作为RT-PCR反应中的扩增靶标，采用所述试剂盒将各洗脱液分别进行同步RT-PCR扩增反应，扩增反应结束后获得不同的Ct值，以黄曲霉毒素浓度的对数值为横坐标，以Ct值为纵坐标，进行回归分析，得到黄曲霉毒素的定量S型标准曲线；
    (2)Nor-1基因转录量RT-PCR标准曲线的建立：将nor-1基因DNA片段Tq-nor1已知拷贝数的样本连续稀释成不同拷贝数，然后采用所述试剂盒对各拷贝数Tq-nor1分别进行同步RT-PCR扩增反应，扩增反应结束后获得不同的Ct值，以Tq-nor1拷贝数的对数值为横坐标，以Ct值为纵坐标，进行回归分析，得到nor-1基因转录量的定量标准曲线；
    (3)培养黄曲霉菌株，取黄曲霉菌孢子进行振荡培养，培养结束后，过滤取菌株培养液和黄曲霉菌丝球；将菌株培养液稀释一定倍数后，替代步骤(1)所述免疫反应中的黄曲霉毒素标品参与免疫竞争反应，免疫竞争反应后将结合在黄曲霉毒素单克隆抗体上的表面展示黄曲霉毒素抗独特型纳米抗体的噬菌体洗脱，将洗脱液中噬菌体释放的DNA分子作为同步RT-PCR扩增反应中定量扩增黄曲霉毒素的模板；另将黄曲霉菌丝球干燥后，提取总RNA并反转录成cDNA，将该cDNA稀释一定倍数后作为同步RT-PCR扩增反应中定量扩增Nor-1基因的模板；
    (4)以所述噬菌体释放的DNA分子和所述cDNA为模板进行同步RT-PCR扩增反应，扩增反应结束后获得两个Ct值，将两个Ct值分别代入黄曲霉毒素的定量S型标准曲线和nor-1基因转录量的定量标准曲线，换算得到黄曲霉毒素浓度和nor-1基因转录量，以此确定黄曲霉毒素产量与Nor-1基因转录量的比值。
  7. 根据权利要求6所述的用来鉴定评价黄曲霉毒素产毒菌株产毒力的方法，其特征在于，所述表面展示黄曲霉毒素抗独特型纳米抗体的噬菌体为噬菌体VHH 2-5，所述黄曲霉毒素单克隆抗体为黄曲霉毒素单克隆抗体1C11。
  8. 根据权利要求6所述的用来鉴定评价黄曲霉毒素产毒菌株产毒力的方法，其特征在于，所述同步RT-PCR扩增反应的反应体系中，上下游引物：Ph-F、Ph-R、Tq-nor1-F、Tq-nor1-R的终浓度均为300～400nM；荧光探针：Ph-probe和Tq-probe的终浓度200～400nM；DNA聚合酶最终用量：0.5U～1.0U；MgCl 2终浓度：1mM～2mM；dNTPs终浓度：200uM～400uM。
  9. 根据权利要求6所述的用来鉴定评价黄曲霉毒素产毒菌株产毒力的方法，其特征在于，所述同步RT-PCR扩增反应的反应体系包括：通用型探针qPCR预混液5μL、Ph-F 0.1μL、Ph-R 0.1μL、Ph-probe 0.1μL、噬菌体模板2μL、Tq-nor1-F 0.1μL、Tq-nor1-R0.1μL、Tq-probe 0.1μL、Nor-1基因模板1μL、DNA聚合酶0.2μL、MgCl 2 0.8μL、dNTPs 0.2μL，加H 2O补齐10μL。
  10. 根据权利要求1所述的用来鉴定评价黄曲霉毒素产毒菌株产毒力的方法，其特征在 于，将所述黄曲霉毒素产毒菌株产毒力定义为Y，将黄曲霉毒素产量与Nor-1基因转录量(AFT/Nor-1)的比值定义为X，建立黄曲霉菌株产毒力的鉴定方程为：Y＝10.14X-16.20，根据高产毒菌株，产毒量>150ng/mL；中产毒：50<产毒量<150ng/mL；低产毒：产毒量<50ng/mL；不产毒：0；代入方程计算得到产毒力鉴定范围：AFT/Nor-1>16.4，为高产毒菌株；6.5<AFT/Nor-1<16.4，为中产毒菌株；0<AFT/Nor-1<6.5，为低产毒菌株；AFT/Nor-1＝0，为不产毒菌株。
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CN113899905A (zh) * 2021-09-07 2022-01-07 中国农业科学院油料作物研究所 一种黄曲霉毒素污染风险的分子预警方法及其应用
CN113899905B (zh) * 2021-09-07 2023-06-27 中国农业科学院油料作物研究所 一种黄曲霉毒素污染风险的分子预警方法及其应用
CN115058444A (zh) * 2022-06-28 2022-09-16 深圳技术大学 一种黄曲霉毒素菌株及其构建方法与应用

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