WO2014067390A1 - 一种针对蚊媒病毒的悬浮芯片多重非诊断性检测引物及方法 - Google Patents

一种针对蚊媒病毒的悬浮芯片多重非诊断性检测引物及方法 Download PDF

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WO2014067390A1
WO2014067390A1 PCT/CN2013/085265 CN2013085265W WO2014067390A1 WO 2014067390 A1 WO2014067390 A1 WO 2014067390A1 CN 2013085265 W CN2013085265 W CN 2013085265W WO 2014067390 A1 WO2014067390 A1 WO 2014067390A1
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primer
virus
microspheres
biotin
pcr
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PCT/CN2013/085265
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French (fr)
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王静
杨宇
李莉
王旺
赵婷婷
孙肖红
刘丽娟
徐宝梁
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Wang Jing
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Priority to ZA2015/02579A priority Critical patent/ZA201502579B/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage

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  • the present invention provides a multi-non-diagnostic detection method for a suspension chip of a mosquito-borne virus. Background technique
  • PCR Polymerase chain reaction
  • the detection method of PCR products is agarose gel electrophoresis. And the agarose gel can only distinguish DNA fragments that differ by about 100 bp. PCR products with little or no fragment length will be incapable of agarose gel electrophoresis.
  • agarose gel electrophoresis is performed by ultraviolet light to excite fluorescent dyes to generate fluorescence, to determine whether or not PCR products are present, and the detection sensitivity is relatively low.
  • the agarose gel electrophoresis judges the product by the size of the fragment, and the specificity is poor, and the fragments of similar size for non-specific amplification cannot be distinguished.
  • dyes commonly used in electrophoresis such as ethidium bromide, are carcinogenic, and frequent exposure is detrimental to health.
  • Mosquito-borne diseases are a group of diseases transmitted by mosquitoes, including yellow fever, dengue fever, Japanese encephalitis, and West Nile fever, which are important public health problems.
  • yellow fever, dengue fever, Japanese encephalitis, and West Nile fever are important public health problems.
  • the incidence of mosquito-borne diseases in the world is on the rise, and the prevalence of the original diseases is expanding and the frequency of diseases is increasing.
  • the corresponding detection and monitoring techniques should be followed up in time to accommodate new disease surveillance needs.
  • the detection and monitoring of pathogens carried by mosquitoes is a method for early monitoring and detection of epidemics in infectious disease surveillance, and is of great significance for effective early warning and control of mosquito-borne diseases.
  • mosquito-borne pathogens involve microbiology and Parasitology specialty.
  • mosquito-borne disease surveillance is also directed to single-species diseases. Different pathogen classification professionals conduct tests from their own professional fields, waste manpower and financial resources, and each professional implements independently, which is not conducive to efficient and comprehensive prevention and control measures. Therefore, the detection of mosquito-borne diseases should be considered comprehensively.
  • the suspension array also known as the liquid chip (Iquid array) is a very flexible and versatile technology platform for biomacromolecule detection, receptor and ligand recognition analysis of proteins, nucleic acids, etc. Wait for research.
  • the suspension chip is mainly used for the qualitative and quantitative analysis of the measured object by the fluorescent coded microspheres and the two lasers which can be coupled to the probe. 100 different biological reactions can be completed in one reaction well, thereby realizing multiple nucleic acids. Detection.
  • the traditional non-diagnostic detection method of the floating chip is time consuming and cumbersome.
  • the selection of the hybridization temperature is also a bottleneck in the development of the technology due to the presence of multiple probes. Summary of the invention
  • an object of the present invention is to provide a multi-non-diagnostic detection method for a suspension chip of a mosquito-borne virus with high sensitivity and specificity.
  • the present invention provides a multi-diagnostic detection method for a floating chip of a mosquito-borne virus, specifically:
  • reaction products are respectively hybridized with the corresponding coding microspheres
  • Anti-Tag on the microsphere specifically captures the Tag sequence on the PCR product under certain conditions
  • primer sequences involved are:
  • the primer upstream primer 5 needs to be manually added with a label and connected by a spacer sequence, and the downstream primer 5 is labeled with biotin.
  • hybridization is carried out by simultaneously adding microspheres, PCR amplification products, SA-PE, at 37 ° C for hybridization, and at the same time, PCR can be performed on streptavidin-phycoerythrin and microspheres. Biotin binding on the product.
  • An improved multi-non-diagnostic detection method for a mosquito chip virus suspension chip comprising the following steps: 1) PCR detection of primer labeling Tag and biotin, 2) PCR amplification of the sample to be tested; 3) adding corresponding microspheres and The PCR product is hybridized; 4) the hybridized microspheres are detected by the machine; wherein, according to the virus to be detected, the specific primer used for the detection is designed, the upstream primer 5 is labeled with different tags, and the primer and the tag are connected with C9 or C18. As a plus arm, the downstream primer 5' is biotinylated for detection. Further, the primer sequences involved are:
  • primer upstream primer 5 needs to be manually added with a label and connected by a spacer sequence, and the downstream primer 5 is labeled with biotin.
  • the hybridization is carried out by simultaneously adding microspheres, PCR amplification products, SA-PE, and the conditions are 37 ° C for hybridization, and at this time, streptavidin-phycoerythrin can be carried out under the conditions. Binding to biotin on the PCR product captured on the microspheres.
  • the invention utilizes the suspension chip system as a platform, combines the multiplex PCR technology and the TAG technology to specifically identify the mosquito vector virus, has high detection sensitivity and good specificity, is a high-throughput mosquito detection, and is a mosquito-borne disease monitoring and the like. It may provide a basis for the detection and early warning of new mosquito-borne pathogens that may be introduced into China.
  • Figure 1 shows the DNA concentration--the fluorescence intensity dose response curve on the surface of the microspheres; the surface fluorescence intensity of the encoded microspheres is positively correlated with the amount of template added, and the abscissa is the template DNA added to the tube during multiplex PCR. The amount, the ordinate represents the fluorescence intensity of the corresponding encoded microsphere surface.
  • Semi-quantitative analysis of nucleic acids can be achieved by fitting a standard curve.
  • Figure 2 shows the method established by the present invention for oriental equine encephalitis virus, western equine encephalitis virus, Venezuelan equine encephalitis virus, chikungunya virus, yellow fever virus, West Nile virus, dengue virus, type B brain
  • the results of detection of the inflammatory virus St. Louis encephalitis virus, Banna virus, Bunia virus, and Rift Valley fever virus.
  • the X axis represents the detection sample
  • the x axis represents the detection microsphere
  • the Z axis represents the detection fluorescence signal (MF I ).
  • the invention discloses a multi-non-diagnostic detection method for a suspension chip of a mosquito-borne virus, and relates to an improvement of reaction conditions and steps in a non-diagnostic detection method of a suspension chip.
  • the chip in the method consists mainly of labeled microspheres, a labelled and biotinylated primer, streptavidin-phycoerythrin, the method comprising: using a labeled upstream primer and a terminal biotin Multiplex PCR of downstream primers to amplify the multiplexed product of the tag, and utilize the specificity of binding between the tags and the uniformity of hybridization temperature The multiplicity of hybridization is achieved. In the hybridization process, the capture of the PCR product by the microspheres is achieved, and the streptavidin-phycoerythrin is combined with the biotin on the PCR product captured on the microsphere, and then the red laser is used.
  • the invention mainly aims to comprehensively detect a plurality of carried pathogens, such as a flavivirus and an alphavirus, for mosquitoes collected in the field.
  • a plurality of carried pathogens such as a flavivirus and an alphavirus
  • High-throughput detection of wild-collected mosquito samples in the same batch, economical and efficient detection of mosquito-borne pathogens, provides simplified detection and monitoring procedures and procedures for the health and disease prevention department to improve efficiency and reduce duplication of effort.
  • the technical solution provided by the invention is a multi-non-diagnostic detection method for a floating chip of a mosquito-borne virus, the method comprising:
  • the specific detection primers were designed according to the nucleotide sequence in the PCR amplification region, and the specificity of the primers was detected by GENEBANK.
  • the 5' end of the upstream primer was ligated with a specific tag, and the downstream primer 5 was subjected to biotin labeling.
  • the reaction products are respectively hybridized with the corresponding coding microspheres; the Ant i-Tag on the microspheres specifically captures the Tag sequence on the PCR product under certain conditions;
  • the streptavidin-phycoerythrin is then added to the biotin on the PCR product captured on the microspheres, and detected by a suspension chip LUM I NAX system, by exciting the red sorting fluorescence on the microsphere matrix and the surface of the microspheres.
  • the specific reaction is combined with phycoerythrin, and the analytes are qualitatively and quantitatively analyzed.
  • the invention also provides an improved multi-diagnostic detection method for a floating chip of a mosquito-borne virus, comprising the following steps: 1. PCR detection of primer labeled Tag and biotin, 2. PCR amplification of the sample to be tested; 3. Adding corresponding The microspheres are hybridized with the PCR product; 4. The hybrid microspheres are detected by the machine.
  • the specific primer used for the detection is designed, the upstream primer 5' marks different tags, the primer and the tag are connected with C9 or C18 as the addition arm, and the downstream primer 5 is used for the biotin. Mark for testing.
  • the method comprises:
  • the B i o-P l exTM system is used to detect the number of microspheres by exciting the red sorting fluorescence on the microsphere matrix; the bound PCR product is quantitatively analyzed by stimulating the phycoerythrin on the surface of the microspheres.
  • the invention Compared with the traditional PCR non-diagnostic detection method, the invention has the following advantages:
  • the detection specificity is much better than the length of the fragment to judge the PCR product.
  • Fragments of similar or identical fragment lengths can also be distinguished and identified, making the design of multiplex PCR primers more flexible.
  • the invention Compared with the conventional non-diagnostic detection method of the floating chip, the invention has the advantages that:
  • TAG technology uniformizes the hybridization temperature, making the hybridization reaction of multiple pathogenic PCR products easier to carry out at the same temperature, increasing the throughput of detection.
  • the TAG sequence is introduced at the 5' end of the upstream primer, and the biotin is labeled at the 5' end of the downstream primer.
  • This design can make the hybridization reaction between the microsphere and the PCR product and the reaction between biotin and streptavidin-phycoerythrin. Simultaneously, the reaction time is saved and the efficiency of detection is improved.
  • the multi-non-diagnostic detection method of the suspension chip of the invention can be used for twelve mosquito-borne viruses: oriental equine encephalitis virus, western equine encephalitis virus, Venezuelan equine encephalitis virus, chikungunya virus, yellow fever virus, west Detection of twelve PCR products of Nile encephalitis virus, dengue virus, Japanese encephalitis virus, St. Louis encephalitis virus, Banna virus, Bunia virus, and Rift Valley fever virus.
  • Bio-PlexTM system detects and quantifies the numbering of the microspheres by activating the red-category fluorescence on the microsphere matrix; the bound PCR product is quantified by stimulating the phycoerythrin on the surface of the microsphere.
  • Dilute the target nucleic acid to be tested generally set more than 6 dilutions, perform PCR reaction with the above PCR program and reaction conditions, and perform PCR product detection with the above reaction.
  • use B i 0-PI ex Ve rsi On the 4.0 analysis system provides the equation fitting standard curve, according to the standard curve, substituting the MFI value of each sample to be tested, can realize the quantitative analysis of the nucleic acid of the sample to be tested.
  • hybridization detection was performed with a PCR negative control as a detection background.
  • the data output by the instrument is the median value of the fluorescence intensity of a numbered microsphere group in the corresponding reaction system ⁇ /ledian Fluorescence Intensity (MFI), which is the number of microspheres read. A statistical average of the signal intensity of each microsphere of the group (100 or more).
  • the fluorescence value (MFI) of each well and the background fluorescence value (Background MFI, BFI) were read by the Bio-Plex suspension chip system.

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Abstract

本发明公开了一种蚊媒病毒的悬浮芯片多重非诊断性检测方法，以及对悬浮芯片非诊断性检测方法中反应条件及步骤的改进。本方法中的芯片主要由带有标签微球、带有标签及生物素化的引物、链霉亲和素一藻红蛋白组成。本发明的方法和芯片可用于检测多种携带的病原体，如黄病毒属病毒、甲病毒属病毒等。

Description

说明书：
一种针对蚊媒病毒的悬浮芯片多重非诊断性检測引物及方法 技术领域
本发明提供一种针对蚊媒病毒的悬浮芯片多重非诊断性检测方 法。 背景技术
聚合酶链反应（Po l yme rase Cha i n React i on ，PCR)是 80年代中 期发展起来的体外核酸扩增技术，也是目前核酸检测的主流技术， PCR 产物的检测判定方法为琼脂糖凝胶电泳，而琼脂糖凝胶仅可区分相差 约 1 00bp的 DNA片段。 片段长度相差不大或者相等的 PCR产物， 琼脂 糖凝胶电泳将无能无力。另外， 琼脂糖凝胶电泳通过紫外光激发荧光 染料产生荧光来判断是否存在 PCR产物， 检测灵敏度相对较低。且琼 脂糖凝胶电泳通过片段的大小对产物进行判断， 特异性差， 对非特异 扩增的大小相似的片段不能区分。而且电泳时常用的染料如溴化乙锭 等具有致癌性， 常接触对身体健康不利。
蚊媒病是一组以蚊虫为媒介而传播的疾病，包括黄热病、登革热、 流行性乙型脑炎、 西尼罗热等，是重要的公共健康问题。 随着全球气 候逐渐上升、 城市化进程的加快、 旅游和贸易的快速发展、 生态环境 的不断变化， 全球蚊媒传染病发病呈上升趋势， 原有疾病的流行区域 不断扩展、 疾病的流行频度不断增加。 随着疾病谱发生变化， 相应的 检测和监测技术要及时跟进， 以适应新的疾病监测需求。检测及监测 蚊虫携带的病原体，在传染病监测中属于早期监测和发现传染病流行 的方法， 对于蚊媒传播疾病的有效预警和控制有重要的意义。 以往的 研究都是针对一种病原体检测，且有些非诊断性检测方法灵敏度不够 高， 不能够早期发现蚊媒传染病的流行； 此外，从生物学分类角度来 看， 蚊媒病原体涉及微生物学与寄生虫学专业， 目前蚊媒病监测也是 针对单病种， 不同病原体分类专业人员分别从自身专业领域进行检 测， 浪费人力、 财力， 且各专业独力实施， 不利于高效、 综合采取防 治措施。 因而， 对于蚊媒病的检测应当综合考虑。 悬浮芯片 (suspension array)也称液相芯片 ( l iquid array, l iquid chip), 是一种非常灵活的多功能技术平台， 可以进行蛋白、 核酸等生物大分子检测、 受体和配体识别分析等研究。悬浮芯片主要 通过可偶联探针的荧光编码微球与两束激光检测，对被测物的定性和 定量分析， 一个反应孔内可以完成 100种不同的生物学反应， 从而实 现对核酸的多重检测。 但传统的悬浮芯片的非诊断性检测方法耗时 长， 步骤繁瑣， 在多重检测中， 由于多重探针的存在， 杂交温度的选 择也是一个技术开发时的瓶颈。 发明内容
针对现有技术存在的问题，本发明的目的在于提供一种检测灵敏 度高， 特异性好的针对蚊媒病毒的悬浮芯片多重非诊断性检测方法。
为实现上述目的，本发明一种针对蚊媒病毒的悬浮芯片多重非诊 断性检测方法， 具体为：
1) 根据 PCR扩增区域内的核苷酸序列设计特异性检测引物， 并 提交 GENEBANK检测引物的特异性， 合成时将上游引物 5' 末端连接 特异 Tag, 下游引物 5， 末端进行生物素标记；
2) PCR反应结束后将反应产物分别与相应的编码微球杂交；
3)微球上的 Anti-Tag在一定的条件下特异的捕获 PCR产物上的 Tag序列；
4) 加入链霉亲和素一藻红蛋白与微球上捕获的 PCR产物上的生 物素结合， 利用悬浮芯片 LUMINAX系统进行检测， 通过激发微球基质 上的红色分类荧光和微球表面经特异性反应结合上的藻红蛋白，对待 检测物进行定性和定量分析；
其中， 所涉及的引物序列为：
Figure imgf000004_0001
VEEV-48-R TGCTATTGGACTATGGCAGCTC 基孔肯亚病毒 CHIK-54-F AGAATTTGCATCAGCATACAGGG
CHIK-54-R TGAAGACATTGGCCCCACAATGAAT 黄热病毒 YF-36-F GAGGTGCATTGGTCTGCAAATCG
YF-36-R GCCCAGGGTTTTTCCCTGAGC 西尼罗脑炎病毒 WN-55-F AGTAGTGTTTGTGAGGATTAACAA
WN-55-R GCGTTTTAGCATATTGACAGCCCG 登革病毒 DEN-33-F TGTAGCTCCGCCAATAATGGGAGG
DEN-33-R TCTGGTCTTTCCCAGCGTCAATA 乙型脑炎病毒 JEV-61-F TTACAGCATTAGCCCCGACCAAGG
JEV-61-R TGATTGAGCCTTCATTTCCTCCTCT 圣路易斯脑炎病毒 SLEV-44-F CAGACCTCCGGAAAGTTGGGGAA
SLEV-44-R CCTCACAACTCCGTTCACCATC 版納病毒 Ban-42-F ATAGAGAATCATCAGTAATTACA
Ban-42-R ATATTACACACTTCATTAACCT 布尼亚病毒 BUN-18-F TCGCATCCACCGGTGCAAATGGATT
BUN-18-R TTATTGACGACCTCCACCTGCCAC 裂谷热病毒 RVF-53-F TGGCATCAGGGCTCGGAAGCA
RVF-53-R ATGGCCTTGGTTCCACTTCCTT 进一步，所述引物上游引物 5，端需要人工添加一段标签并且之间 用间隔序列相连， 下游引物 5，端经生物素标记。
进一步， 所述杂交中同时加入微球， PCR扩增产物， SA-PE , 条 件为 37°C进行杂交， 同时此条件下可以进行链霉亲和素一藻红蛋白 与微球上捕获的 PCR产物上的生物素结合。
一种改进的针对蚊媒病毒的悬浮芯片多重非诊断性检测方法，包 括以下步骤： 1 ) PCR检测引物标记 Tag和生物素， 2 ) PCR扩增待检 样品； 3 ) 加入相应的微球与 PCR产物杂交； 4 ) 上机对杂交后的微球 进行检测； 其中， 根据待检测的病毒， 设计检测所用的特异引物，上 游引物 5， 标记不同的 Tag,引物与 Tag之间连接 C9或 C18作为加臂， 下游引物 5 ' 进行生物素标记， 以便检测。 进一步， 所涉及的引物序列为:
Figure imgf000006_0001
进一步，所述引物上游引物 5，端需要人工添加一段标签并且之间 用间隔序列相连， 下游引物 5，端经生物素标记。
进一步， 所述杂交中同时加入微球， PCR扩增产物， SA-PE , 条 件为 37°C进行杂交， 同时此条件下可以进行链霉亲和素一藻红蛋白 与微球上捕获的 PCR产物上的生物素结合。
本发明利用悬浮芯片系统作为平台，结合多重 PCR技术及 TAG技 术可以蚊媒病毒进行特异性的识别， 检测灵敏度高， 特异性好， 是一 种高通量的蚊虫检测，为蚊媒病监测以及可能传入我国的新型蚊媒病 病原体的检测以及预警提供了基础。 附图说明
图 1 为 DNA浓度——编码微球表面的荧光强度剂量反应曲线；编 码微球表面荧光强度与加入的模板量在一定的范围内成正相关，其中 横坐标为多重 PCR时管中加入的模板 DNA的量，纵坐标代表相应编码 微球表面的荧光强度。可以通过拟合的标准曲线实现对核酸的半定量 分析。
图 2表示用本发明建立的方法对东方马脑炎病毒、西方马脑炎病 毒、 委内瑞拉马脑炎病毒、 基孔肯亚病毒、 黄热病毒、 西尼罗病毒、 登革病毒、 乙型脑炎病毒、 圣路易斯脑炎病毒、 版納病毒、 布尼亚病 毒、 裂谷热病毒进行检测的结果。 其中， X轴表示检测样品， 丫轴表 示检测微球， Z轴表示检测荧光信号 （MF I )。 具体实施方式
下面， 参考附图， 对本发明进行更全面的说明， 附图中示出了本 发明的示例性实施例。 然而， 本发明可以体现为多种不同形式， 并不 应理解为局限于这里叙述的示例性实施例。 而是， 提供这些实施例， 从而使本发明全面和完整，并将本发明的范围完全地传达给本领域的 普通技术人员。 本发明公开了本发明提供一种蚊媒病毒的悬浮芯片多重非诊断 性检测方法，还涉及对悬浮芯片非诊断性检测方法中反应条件及步骤 的改进。 本方法中的芯片主要由带有标签微球、 带有标签及生物素化 的引物、链霉亲和素一藻红蛋白组成， 所述方法包括： 利用带有标签 的上游引物和末端生物素化的下游引物多重 PCR扩增出带有标签的 多重目的产物，并且利用标签之间结合的特异性以及杂交温度的统一 性实现杂交的多重性， 杂交过程中既实现了微球对 PCR产物的捕获， 同时完成链霉亲和素一藻红蛋白与微球上捕获的 PCR产物上的生物 素结合， 之后以红色激光激发其球形基质上的分类荧光， 依据其球形 基质色彩不同确定类型； 以绿色激光激发藻红蛋白， 测定球形基质上 结合的报告荧光分子的数量，用于间接确定球形基质上结合的 PCR产 物的含量。
本发明主要针对野外采集的蚊虫综合检测多种携带的病原体，如 黄病毒属病毒、 甲病毒属病毒等。将野外采集蚊虫样本同批次处理后 高通量检测， 经济、 高效检测蚊传病原体， 可为卫生防病部门提供简 化的检测、 监测步骤和程序， 提高效率和减少重复工作量。
本发明提供的技术方案是一种针对蚊媒病毒的悬浮芯片多重非 诊断性检测方法， 该方法包括：
首先根据 PCR扩增区域内的核苷酸序列设计特异性检测引物，并 提交 GENEBANK检测引物的特异性， 合成时将上游引物 5 ' 末端连接 特异 Tag , 下游引物 5， 末端进行生物素标记；
PCR反应结束后将反应产物分别与相应的编码微球杂交； 微球上的 Ant i -Tag在一定的条件下特异的捕获 PCR产物上的 Tag 序列；
然后加入链霉亲和素一藻红蛋白与微球上捕获的 PCR产物上的 生物素结合， 利用悬浮芯片 LUM I NAX系统进行检测， 通过激发微球基 质上的红色分类荧光和微球表面经特异性反应结合上的藻红蛋白，对 待检测物进行定性和定量分析。
本发明还提供一种改进的针对蚊媒病毒的悬浮芯片多重非诊断 性检测方法， 包括以下步骤： 1、 PCR检测引物标记 Tag和生物素， 2、 PCR扩增待检样品； 3、 加入相应的微球与 PCR产物杂交； 4.上机对 杂交后的微球进行检测。
在本发明的方法中， 首先根据待检测的病毒， 设计检测所用的特 异引物，上游引物 5 ' 标记不同的 Tag,引物与 Tag之间连接 C9或 C18 作为加臂， 下游引物 5， 进行生物素标记， 以便检测。
在本发明的加入相应的微球与 PCR产物杂交的过程中， 包括：
1 . 取带有 Ant i -Tag标签的微球每种各 3500个加入到杂交液 中， 使其总量为 35 μ Ι， 根据微球计数结果计算相应加入量；
2. 向各管中加 5〜1 7u l 各种蚊媒病毒的 PCR产物吹打混匀。
3. 再向各孔加 4ng/u I SA-PE的 1 X TMAC液， 使之总体积变为 80 u l o
4. 37°C下杂交一定时间。
5. 转移至滤板抽滤去掉未结合的 PCR产物再向各孔加入 80u l 1 X TE溶液， 振荡使微球重悬。
6. 反应结束后上机检测。 B i o-P l exTM system进行检测， 通 过激发微球基质上的红色分类荧光， 对微球的编号进行识别；通过激 发微球表面的藻红蛋白， 对结合的 PCR产物进行定量分析。
本发明与传统的 PCR非诊断性检测方法相比， 优点在于：
1 . 通过仪器的信号放大系统和生物素亲和素的放大作用， 本 发明对 PCR产物的检测灵敏度高于琼脂糖凝胶电泳。
2. 通过特异性探针捕获 PCR产物，检测特异性远好于用片段长 度对 PCR产物进行判断。
3. 对片段长度大小相似或者一样的片段， 同样可以进行区分 识别， 从而使多重 PCR引物的设计趋于灵活。
本发明与传统的悬浮芯片非诊断性检测方法相比， 优点在于：
1 . TAG技术的应用， 均一了杂交温度， 使多重病原 PCR产物的杂 交反应更易于在同一温度下进行， 提高了检测的通量。
2. 引物合成时上游引物的 5 ' 端引入 TAG序列，下游引物 5 ' 端标 记生物素， 此设计可以使微球与 PCR产物的杂交反应和生物素与链亲 和素-藻红蛋白的反应同时进行， 节省了反应时间， 提高了检测的效 率。
本发明的悬浮芯片多重非诊断性检测方法，可用于十二种蚊媒病 毒： 东方马脑炎病毒、 西方马脑炎病毒、 委内瑞拉马脑炎病毒、 基孔 肯亚病毒、 黄热病毒、 西尼罗脑炎病毒、 登革病毒、 乙型脑炎病毒、 圣路易斯脑炎病毒、版納病毒、布尼亚病毒、裂谷热病毒的十二重 PCR 产物的检测。
1 .确定上述十二种病毒的诊断片段，通过 NCB I的 GeneBank获取候 选基因序列， 利用软件设计特异检测引物， 并合成相关引物序列，上 游引物 5 ' 标记 Tag, Tag与引物间由 C9或 C18连接， 结果一致； 下游引 物 5， 端生物素标记， 见表 1， 并将这些引物稀释成 10 mo l /L。
表 1 引物序列
Figure imgf000010_0001
2.使用以上引物的多重 PCR反应扩增以上十二种待检测病原。 模拟检测了共 90份采集的蚊样本； 检测荧光值大于等于三倍本 底荧光值判为阳性， 其中检测出登革 3份， 乙脑 4份， 与分离培养以 及荧光定量 PCR结果一致， 样本全部正确识别。
捕获探针对待检测的 PCR产物的捕获和检测：
i . 取带有 Anti-Tag标签的微球每种各 3500个加入到杂交液中， 使其总量为 35 μ I， 根据微球计数结果计算相应加入量；
i i. 向各管中加 5〜17ul 各种蚊媒病毒的 PCR产物吹打混匀。
i i i . 再向各孔加 4ng/u I SA-PE的 1 X TMAC液， 使之总体积变为 80 u I o
iv. 37°C下杂交一定时间。
v. 转移至滤板抽滤去掉未结合的 PCR产物再向各孔加入 80ul 1 X TE溶液， 振荡使微球重悬。
vi. 反应结束后上机检测。 Bio-PlexTM system进行检测， 通过激 发微球基质上的红色分类荧光， 对微球的编号进行识别； 通过激发微 球表面的藻红蛋白， 对结合的 PCR产物进行定量分析。
定量检测：
待检目标核酸系列稀释， 一般设 6个以上稀释度， 同上面的 PCR 程序和反应条件进行 PCR反应， 同上面的反应进行 PCR产物的检测， 根据检测结果， 运用 B i 0-P I ex Ve r s i on 4.0分析系统提供的方程拟 合标准曲线， 根据标准曲线， 代入各个待测样品的 MFI值， 即可实现 待检样品核酸的定量分析。
如登革病毒基因组反转录后 10倍系列稀释， 2.8fg〜280ng， 按 确定的方法进行 PCR扩增和检测， 拟合曲线， 曲线动态范围 28fg〜
280pg, 曲线方程： FI = -25.2499 + (663.894 + 25.2499) / ((1 +
(Cone / 20599.2) 4.5687)^0.0999957， 从标准曲线推算检出限为
50fg/testo 样品编号 空白 1 2 3 4 5 6 浓度 b I ank 2.8 28 280 2800 28000 280000
(fg/test)
MFI 59.5 55.5 209.5 307 874 2127 2236 结果分析:
检测时， 同时以 PCR阴性对照进行杂交检测做为检测本底。对于 每个检测体系和检测本底，仪器输出的数据是相应反应体系内一种编 号微球群的荧光强度中位值 ί/ledian Fluorescence Intensity, MFI ), 亦即读取的这种编号的微球群 （100个或以上） 的每个微球信号强度 的统计平均值。 通过 Bio-Plex悬浮芯片系统读取各孔荧光值 （MFI) 以及本底荧光值 (Background MFI,BFI)。
结果判断：
当待检测样本的 MFI值为本次检测背景信号强度三倍以上时即 判为阳性。

Claims

权利要求书：
1. 一种针对蚊媒病毒的悬浮芯片多重非诊断性检测的引物， 其引物 序列为：
Figure imgf000013_0001
2. 采用权利要求 1所述的引物针对蚊媒病毒的悬浮芯片多重非诊断 性检测方法， 其特征在于， 该方法具体为：
1 ) 根据 PCR扩增区域内的核苷酸序列设计特异性检测引物， 并 提交 GENEBANK检测引物的特异性， 合成时将上游引物 5 ' 末端连接 特异 Tag, 下游引物 5， 末端进行生物素标记；
2 ) PCR反应结束后将反应产物分别与相应的编码微球杂交；
3 )微球上的 Ant i -Tag在一定的条件下特异的捕获 PCR产物上的 Tag序列；
4) 加入链霉亲和素一藻红蛋白与微球上捕获的 PCR产物上的生 物素结合， 利用悬浮芯片 LUM I NAX系统进行检测， 通过激发微球基质 上的红色分类荧光和微球表面经特异性反应结合上的藻红蛋白，对待 检测物进行定性和定量分析。
3. 如权利要求 2所述的非诊断性检测方法， 其特征在于， 所述引物 上游引物 5，端需要人工添加一段标签并且之间用间隔序列相连，下游 引物 5，端经生物素标记。
4. 如权利要求 2所述的非诊断性检测方法， 其特征在于， 所述杂交 中同时加入微球， PCR扩增产物， SA-PE, 条件为 37°C进行杂交， 同 时此条件下可以进行链霉亲和素一藻红蛋白与微球上捕获的 PCR产 物上的生物素结合。
5. 一种改进的针对蚊媒病毒的悬浮芯片多重非诊断性检测方法， 其 特征在于， 所述方法包括以下步骤： 1 ) PCR检测引物标记 Tag和生 物素， 2) PCR扩增待检样品； 3) 加入相应的微球与 PCR产物杂交； 4) 上机对杂交后的微球进行检测； 其中， 根据待检测的病毒， 设计 检测所用的特异引物，上游引物 5， 标记不同的 Tag,引物与 Tag之间 连接 C9或 C18作为加臂， 下游引物 5， 进行生物素标记， 以便检测。
6. 如权利要求 5所述的非诊断性检测方法， 其特征在于， 所述非诊 断性检测方法涉及的引物序列为：
Figure imgf000014_0001
委内瑞拉马脑炎病毒 VEEV-48-F ACGACAGAAAACCAGCAGAGACTT
VEEV-48-R TGCTATTGGACTATGGCAGCTC 基孔肯亚病毒 CHIK-54-F AGAATTTGCATCAGCATACAGGG
CHIK-54-R TGAAGACATTGGCCCCACAATGAAT 黄热病毒 YF-36-F GAGGTGCATTGGTCTGCAAATCG
YF-36-R GCCCAGGGTTTTTCCCTGAGC 西尼罗脑炎病毒 WN-55-F AGTAGTGTTTGTGAGGATTAACAA
WN-55-R GCGTTTTAGCATATTGACAGCCCG 登革病毒 DEN-33-F TGTAGCTCCGCCAATAATGGGAGG
DEN-33-R TCTGGTCTTTCCCAGCGTCAATA 乙型脑炎病毒 JEV-61-F TTACAGCATTAGCCCCGACCAAGG
JEV-61-R TGATTGAGCCTTCATTTCCTCCTCT 圣路易斯脑炎病毒 SLEV-44-F CAGACCTCCGGAAAGTTGGGGAA
SLEV-44-R CCTCACAACTCCGTTCACCATC 版納病毒 Ban-42-F ATAGAGAATCATCAGTAATTACA
Ban-42-R ATATTACACACTTCATTAACCT 布尼亚病毒 BUN-18-F TCGCATCCACCGGTGCAAATGGATT
BUN-18-R TTATTGACGACCTCCACCTGCCAC 裂谷热病毒 RVF-53-F TGGCATCAGGGCTCGGAAGCA
RVF-53-R ATGGCCTTGGTTCCACTTCCTT
7. 如权利要求 6所述的非诊断性检测方法， 其特征在于， 所述引物 上游引物 5，端需要人工添加一段标签并且之间用间隔序列相连，下游 引物 5，端经生物素标记。
8. 如权利要求 6所述的非诊断性检测方法， 其特征在于， 所述杂交 中同时加入微球， PCR扩增产物， SA-PE, 条件为 37°C进行杂交， 同 时此条件下可以进行链霉亲和素一藻红蛋白与微球上捕获的 PCR产 物上的生物素结合。
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