WO2014153948A1 - 针对蜱传病原的悬浮芯片多重检测引物及方法 - Google Patents

针对蜱传病原的悬浮芯片多重检测引物及方法 Download PDF

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WO2014153948A1
WO2014153948A1 PCT/CN2013/085199 CN2013085199W WO2014153948A1 WO 2014153948 A1 WO2014153948 A1 WO 2014153948A1 CN 2013085199 W CN2013085199 W CN 2013085199W WO 2014153948 A1 WO2014153948 A1 WO 2014153948A1
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primer
microspheres
biotin
detection method
pcr
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French (fr)
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王静
杨宇
李莉
刘丽娟
刘键
王旺
赵婷婷
徐宝梁
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Wang Jing
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Priority to RU2015143410A priority Critical patent/RU2633692C2/ru
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6893—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for protozoa
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the present invention relates to a multi-non-diagnostic detection method for a suspension chip for sputum-borne pathogens. Background technique
  • PCR Polymerase chain reaction
  • the detection method of PCR products is agarose gel electrophoresis.
  • 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.
  • Earthworm is a blood-sucking parasite that is parasitic on the surface of animals. As one of the most important medical media, it is a variety of species, widely distributed, and diverse in life. It can invade a variety of hosts such as birds, reptiles, and mammals. It can spread a variety of pathogens, most of which are important natural epidemic diseases and zoonotic diseases, and most of them do not have effective vaccines, which poses a major hazard to human health and animal husbandry. The study of non-diagnostic methods for rumor-borne diseases should be further strengthened, and the establishment of a multi-non-diagnostic test method for simple, effective, highly sensitive and highly specific rumor diseases has become a necessary necessity.
  • rumored pathogens involve bacteria and viruses. And a variety of microbiology such as rickettsia, spirochete, etc.
  • most of the rumored pathogen detection is only for a single pathogen, and different pathogen classification professionals are testing from their own professional fields, wasting human and financial resources, and each professional is independent. Implementation is not conducive to efficient and comprehensive prevention and control measures. Therefore, the detection of rumored pathogens should be comprehensively considered.
  • Suspensing chip (suspens i on ar ray) is also called liquid chip (li qu id ar ray, l iquid chip), is a very flexible and versatile technology platform for biomacromolecule detection, receptor and ligand recognition analysis of proteins and nucleic acids.
  • 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
  • the present invention provides a multi-diagnostic detection method for a suspended chip of a sputum-transmitting pathogen, 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 upstream primer 5 of the primer 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 suspended chip for sputum pathogens comprising the following steps:
  • the specific primer used for the detection is designed, the upstream primer 5, the end is connected with a specific tag, and the primer and the tag are connected with C9 or C18 as a plus arm, and the downstream primer 5 is at the end for biotin labeling.
  • primer sequences involved are:
  • the upstream primer 5 of the primer 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.
  • amplification conditions are:
  • the invention utilizes the suspension chip system as a platform, combines the multiplex PCR technology and the TAG technology to carry out specific recognition of pathogens, has high detection sensitivity and good specificity, and is a high-throughput non-diagnostic detection method, which is a rumor. Disease surveillance and possible introduction to our country The basis for the detection and early warning of new sputum-transmitting pathogens. DRAWINGS
  • Figure 1 is a DNA concentration-encoded fluorescence intensity dose response curve on the surface of the microsphere; the surface fluorescence intensity of the encoded microsphere is positively correlated with the amount of template added, wherein 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 forest encephalitis virus, Xinjiang hemorrhagic fever virus, spotted fever group rickettsia, Babesi protozoa, Erik body, Tularia serrata, Q rickettsia, The results of detection by Borrelia burgdorferi and Bartonella; wherein the X axis represents the detection sample, the ⁇ axis represents the detection microsphere, and the Z axis represents the detection fluorescence signal (MF I ).
  • the invention provides a multi-non-diagnostic detection method for a suspension chip of a sputum-transmitting pathogen, and relates to a non-diagnostic detection method for a suspension chip and an improvement of reaction conditions and steps in a PCR process, in particular, detection of a merging primer into 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
  • the multiplex PCR of the downstream primers amplified the multiplexed product of the tag, and the multiplicity of the hybridization was achieved by the specificity of the binding between the tags and the uniformity of the hybridization temperature.
  • the hybridization process realized both the microspheres and the PCR products.
  • the TAG technology tag is composed of only three bases and cannot be combined with any natural DNA and PCR products, thereby ensuring the specificity of the hybridization process and the uniformity of the conditions, and at the same time making the detection process simpler and more efficient.
  • the invention mainly aims at comprehensive detection of a variety of pathogens carried in the field, such as forest encephalitis virus, Q rickettsia, and the like.
  • High-throughput detection of field-collected aphid samples in the same batch, economical and efficient detection of rumored pathogens provides simplified detection, monitoring procedures and procedures for the health and disease prevention department to improve efficiency and reduce duplication of effort. It can also be used for rapid detection of pathogens carried by import and export, border items, food, etc.
  • the invention provides a multi-non-diagnostic detection method for a suspension chip of a sputum-borne pathogen, 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 primer upstream primer 5 used in the method needs to manually add a segment of the tag and is 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, the PCR products captured on the streptavidin-phycoerythrin and the microspheres can be carried out. Biotin combination.
  • the present invention also provides a multiplex PCR method capable of efficiently amplifying nine ruminant pathogens, the method comprising the steps of:
  • the pathogens of the corresponding genus can be tested positive, and some pathogen primers are designed as merging primers; 3.
  • the upstream primer 5 is connected to the specific tag at the end, and the primer and the tag are connected to C9 or C18 as the addition arm, and the downstream primer 5 is labeled with biotin.
  • the invention also provides an improved multi-non-diagnostic detection method for a suspended chip of a sputum-transmitting path, comprising the following steps: 1. PCR amplifying a sample to be tested; 2. adding a corresponding microsphere to hybridize with the PCR product; The machine detects the hybridized microspheres.
  • Amplification conditions 95 ° C 5 m i n
  • the method comprises the following steps: 1. Adding 3500 microspheres each with an Ant i-Tag tag to the hybridization solution to make the total amount of 35 ⁇ I, calculating the corresponding addition amount according to the microsphere counting result;
  • the B i o-P l exTM system performs detection to identify the number of the microspheres by exciting the red sorting fluorescence on the microsphere matrix; and quantitatively analyzing the bound PCR products by exciting 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 sensitivity of the PCR product is higher than that of agarose gel electrophoresis by the signal amplification system of the instrument and the amplification of biotin avidin;
  • discrimination can also be made to make the design of multiplex PCR primers more flexible.
  • 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 introduction of a merging base can make the detection range not limited to species and a diseased strain, and can apply the multiplex detection system to the detection of species.
  • the present invention relates to a multi-non-diagnostic detection method for a sputum-borne pathogen suspension chip, which is used for forest encephalitis virus, Xinjiang hemorrhagic fever virus, spotted fever group rickettsia, Babexi original Detection of nine-plex PCR products of worm, Eric, T. serrata, Q rickettsia, Borrelia burgdorferi, Bartonella:
  • the reaction is checked by the B io-P l exTM system after the beam, and the number of the microspheres is recognized by exciting the red-category fluorescence on the microsphere matrix; Phycoerythrin, quantitative analysis of bound PCR products.
  • 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 4. 0 analysis system provides the equation According to the standard curve, according to the standard curve, the MFI value of each sample to be tested can be substituted to realize the quantitative analysis of the nucleic acid of the sample to be tested.
  • hybridization detection using a PCR negative control was also used as a detection background.
  • the data output by the instrument is the median Fluorescence Intensity (MFI) of a number of microspheres in the corresponding reaction system, that is, the number of microspheres read. A statistical average of the signal intensity of each microsphere (100 or more).
  • MFI median Fluorescence Intensity
  • BFI background fluorescence

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Abstract

本发明公开了一种蜱传病原的悬浮芯片多重检测方法，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 PC R 产物的捕获，同时完成链霉亲和素-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；并用于间接确定球形基质上结合的 PCR 产物的含量，本发明可为卫生防病部门提供简化的检测

Description

针对婢传病原的悬浮芯片多重检测引物及方法 技术领域
本发明涉及一种针对埤传病原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 ymerase 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 100bp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。
埤是一种寄生在动物体表的吸血寄生虫，作为最重要的医学媒介 之一， 埤种类多、 分布广、 生活习性多样， 能侵袭鸟类、 爬行类、哺 乳动物等多种宿主， 并且能传播多种病原体， 其中大多数是重要的自 然疫源性疾病和人兽共患病， 且大部分都没有有效的疫苗， 给人类健 康及畜牧业带来重大危害。应进一步加强对埤传疾病的非诊断性检测 方法的研究， 而一种简便有效， 高灵敏高特异的埤传疾病多重非诊断 性检测方法的建立也成为了目前必要之需。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 现埤传传染病的流行； 此外， 从生物学分类角度来看， 埤传病原体涉 及细菌、 病毒及立克次体、 螺旋体等多种微生物学， 目前埤传病原检 测大多仅是针对单种致病原，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， 对于埤传病原的检测应当综合多重考虑。
悬浮芯片 (suspens i on ar ray)也称液相芯片 ( l i qu i d ar ray, 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， 特异性好的针对埤传病原的悬浮芯片多重非诊断性检测方法。
为实现上述目的，本发明一种针对埤传病原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
巴尔通体 Barton-20-f GCTATGTCTGCATTCTATCA
Barton-20-r GATCYTCAATCATTTCTTTCCA 森林 炎病毒 TBEV-36-f: ACAGACTTGARATGGCCATGTGGAG
TBEV-36-r CCCATCACTCCTGTGTCACACT 新疆出血热病毒 XHF-61-F2 TGGACACYTTCACAAACTC
XHF-61-R3 GACAAATTCCCTGCACCA 斑点热群立克次体 SF-54-f GGCAATTAATATCGCTGACGG
SF-54-r GCATCTGCACTAGCACTTTC 巴贝西原虫 Babesia-53- f CTTAGTATAAGCTTTTATACAGC
Babesia-53- r ATAGGTCAGAAACTTGAATGATACA 埃里克体 Ehrlichia-42-F CAATTGCTTATAACCTTTTGGT
Ehrlichia-42-R CCCTATTAGGAGGGATACGACCTT 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。
一种改进的针对埤传病原的悬浮芯片多重非诊断性检测方法，包 括以下步骤：
1 ) PCR扩增待检样品；
2 ) 加入相应的微球与 PCR产物杂交；
3 ) 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， 末端连接特异 Tag, 引物与 Tag之间连接 C9或 C18作为加臂， 下 游引物 5， 末端进行生物素标记。
进一步， 所涉及的引物序列为:
Figure imgf000005_0001
Q-44-r TGGAAGTTATCACGCAGTTG 伯氏疏螺旋体 Bo-33-f ATGCACATCTGGTGTTAACTA
Bo-33-r GACTTATCACCGGCAGTCTTA 巴尔通体 Barton-20-f GCTATGTCTGCATTCTATCA
Barton-20-r GATCYTCAATCATTTCTTTCCA 森林 炎病毒 TBEV-36-f: ACAGACTTGARATGGCCATGTGGAG
TBEV-36-r CCCATCACTCCTGTGTCACACT 新疆出血热病毒 XHF-61-F2 TGGACACYTTCACAAACTC
XHF-61-R3 GACAAATTCCCTGCACCA 斑点热群立克次体 SF-54-f GGCAATTAATATCGCTGACGG
SF-54-r GCATCTGCACTAGCACTTTC 巴贝西原虫 Babesia-53- f CTTAGTATAAGCTTTTATACAGC
Babesia-53- r ATAGGTCAGAAACTTGAATGATACA 埃里克体 Ehrlichia-42-F CAATTGCTTATAACCTTTTGGT
Ehrlichia-42-R CCCTATTAGGAGGGATACGACCTT 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。
进一步， 所述扩增条件为：
95°C 5m i n
95°C 40sec
52°C 40sec
72°C 1 m i n 35个循环
72°C 10m i n
4°C
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 术可以埤传病原体进行特异性的识别， 检测灵敏度高， 特异性好，是 一种高通量的非诊断性检测方法，为埤传疾病监测以及可能传入我国 的新型埤传播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森林脑炎病毒、 新疆出血热病 毒、 斑点热群立克次体、 巴贝西原虫、 埃里克体、 土拉弗朗西斯菌、 Q热立克次体、 伯氏疏螺旋体、 巴尔通体进行检测的结果； 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。
本发明提供一种针对埤传病原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以及 PCR过程中反应条件及 步骤的改进， 尤其是兼并引物引入悬浮芯片的检测。 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 -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。 本发明中使用 TAG技术标签只有三种碱基构成，不能与任何天然 DNA及 PCR产物结合，因此保证了杂交过程的特异性及条件的均一性， 同时使检测过程变得更简捷， 高效。 本发明主要针对野外捕获的埤虫 综合检测多种携带的病原体，如森林脑炎病毒， Q热立克次体等。 将 野外采集埤虫样本同批次处理后高通量检测， 经济、 高效检测埤传病 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。 还可用于进出口、 边境的物品、食品等所携带 的病原体进行快速检测。
本发明一种针对埤传病原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。
该方法使用的引物上游引物 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。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。
本发明还提供一个能有效扩增 9种埤传病原体的多重 PCR方法， 该方法包括以下步骤：
1、 根据 PCR扩增区域内的核苷酸序列设计特异性检测引物， B I 0ED I T软件比对并提交 GENEBANK检测引物的特异性；
2、 为使检测达到更好的兼并性， 对相应属的病原均能有检测阳 性， 部分病原引物设计为兼并引物； 3、 合成时将上游引物 5， 末端连接特异 Tag, 引物与 Tag之间连 接 C9或 C18作为加臂， 下游引物 5， 末端进行生物素标记；
4、 按优化后的配比配制 PCR反应体系并且在优化的条件下进行 扩增。
本发明还提供一种改进的针对埤传病原的悬浮芯片多重非诊断 性检测方法， 包括以下步骤： 1、 PCR扩增待检样品； 2、 加入相应的 微球与 PCR产物杂交； 3、 上机对杂交后的微球进行检测。
在本发明的 PCR扩增过程中， 包括优化体系和优化的扩增条件： 扩增条件： 95°C 5m i n
95°C 40sec 35个循环
Figure imgf000009_0001
4°C o
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 I， 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 各种以埤传病原 DNA为模板的 PCR产物 吹打混匀。
3、 再向各孔加 4ng/u l 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。
本发明与传统的多重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。
3. 兼并碱基的引入可以使检测范围不仅限于种及某病株， 能将 本多重检测体系应用于种属的检测。
如图 1、 图 2所示， 本发明一种针对埤传病原的悬浮芯片多重非 诊断性检测方法， 用于森林脑炎病毒、 新疆出血热病毒、 斑点热群立 克次体、 巴贝西原虫、 埃里克体、 土拉弗朗西斯菌、 Q热立克次体、 伯氏疏螺旋体、 巴尔通体的九重 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 1 0 mo l /L。
表 1 引物序列
Figure imgf000010_0001
TBEV-36-r CCCATCACTCCTGTGTCACACT 新疆出血热病毒 XHF-61-F2 TGGACACYTTCACAAACTC
XHF-61-R3 GACAAATTCCCTGCACCA 斑点热群立克次体 SF-54-ompBf GGCAATTAATATCGCTGACGG
SF-54-ompBr GCATCTGCACTAGCACTTTC 巴贝西原虫 Babesia-53-BAB-f CTTAGTATAAGCTTTTATACAGC
Babesia-53-BAB-r ATAGGTCAGAAACTTGAATGATACA 埃里克体 Ehrlichia-42-HMElF CAATTGCTTATAACCTTTTGGT
Ehrlichia-42-HME3R CCCTATTAGGAGGGATACGACCTT
2、 使用以上引物的多重 PCR反应扩增以上九种待检测病原。
模拟检测了共 100份采集的埤虫样本；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 Q热立克次体 1份， 森林脑炎病毒 2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 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 i i . 向各管中加 5〜1 7u l 各种埤传病原的 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 l o
i v. 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 80u l 1 X TE溶液， 振荡使微球重悬。
v i . 反应 由束后上机检 B i o—P l exTM system进行检 ¾'】， 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 另' J ; 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。
如伯氏疏螺旋体基因组 10倍系列稀释， 8fg〜800ng， 按确定的 方法进行 PCR扩增和检测， 拟合曲线， 曲线方程： Std. Curve: Fl =
-6.53652 + (1881.23 + 6.53652) / ((1 + (Cone /
14.9405) Λ-1.61702) ) ^Ο.916998， 从标准曲线推算检出 限为
173fg/test。
Figure imgf000012_0001
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 Median 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. 一种针对埤传病原的悬浮芯片多重非诊断性检测的引物， 其序列 为：
Figure imgf000013_0001
2. 采用权利要求 1所述的引物针对埤传病原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 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. 一种改进的针对埤传病原的悬浮芯片多重非诊断性检测方法，其 特征在于， 所述非诊断性检测方法包括以下步骤：
1 ) PCR扩增待检样品；
2 ) 加入相应的微球与 PCR产物杂交；
3 ) 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， 末端连接特异 Tag, 引物与 Tag之间连接 C9或 C1 8作为加臂， 下 游引物 5， 末端进行生物素标记。
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 断性检测方法所涉及的引物序列为：
Figure imgf000014_0001
TBEV-36-r CCCATCACTCCTGTGTCACACT 新疆出血热病毒 XHF-61-F2 TGGACACYTTCACAAACTC
XHF-61-R3 GACAAATTCCCTGCACCA 斑点热群立克次体 SF-54-f GGCAATTAATATCGCTGACGG
SF-54-r GCATCTGCACTAGCACTTTC 巴贝西原虫 Babesia-53- f CTTAGTATAAGCTTTTATACAGC
Babesia-53- r ATAGGTCAGAAACTTGAATGATACA 埃里克体 Ehrlichia-42F CAATTGCTTATAACCTTTTGGT
Ehrlichia-42-R CCCTATTAGGAGGGATACGACCTT
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 5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。
9. 如权利要求 5所述的非诊断性检测方法， 其特征在于， 所述扩增 条件为：
95 C 5m i n
95°C 40sec
52°C 40sec
72°C 1 m i n > - 35个循环
72°C 10m i n
4°C
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CN105483232A (zh) * 2015-12-24 2016-04-13 四川国际旅行卫生保健中心 一种立克次体液相基因芯片的检测方法
CN113444773B (zh) * 2021-08-17 2023-06-16 黑龙江省牡丹江林业中心医院 一种基于液相芯片检测蜱虫病原体核酸的方法及试剂盒
CN114058716A (zh) * 2021-12-08 2022-02-18 河套学院 蜱媒病原体多样性的分析方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101560558A (zh) * 2009-03-04 2009-10-21 中国检验检疫科学研究院 多重pcr产物的悬浮芯片检测方法
CN102943128A (zh) * 2012-10-29 2013-02-27 中国检验检疫科学研究院 一种针对蚊媒病毒的悬浮芯片多重非诊断性检测方法
CN103173567A (zh) * 2013-03-27 2013-06-26 中国检验检疫科学研究院 一种针对蜱传病原的悬浮芯片多重非诊断性检测方法

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2062470C1 (ru) * 1992-07-06 1996-06-20 Омский научно-исследовательский институт природноочаговых инфекций ГК санитарно-эпидемиологического надзора РФ Способ диагностики клещевого энцефалита
RU2233888C1 (ru) * 2002-12-15 2004-08-10 Дальневосточный государственный медицинский университет Способ обнаружения микроорганизма "montezuma"
CN101560557B (zh) * 2009-03-04 2012-07-04 中国检验检疫科学研究院 一种联合检测五种烈性病原菌的基因液相芯片及其非诊断性检测方法
WO2010126771A1 (en) * 2009-04-27 2010-11-04 University Of Miami Systems, kits and methods of identifying ocular fungal and amoebic pathogens
RU2415947C1 (ru) * 2009-10-13 2011-04-10 Закрытое акционерное общество "Научно-производственная фирма ДНК-Технология" НАБОР СИНТЕТИЧЕСКИХ ОЛИГОНУКЛЕОТИДОВ ДЛЯ ВЫЯВЛЕНИЯ ДНК ВОЗБУДИТЕЛЯ ЧЕЛОВЕЧЕСКОГО МОНОЦИТАРНОГО ЭРЛИХИОЗА Ehrlichia spp. МЕТОДОМ ПОЛИМЕРАЗНОЙ ЦЕПНОЙ РЕАКЦИИ "В РЕАЛЬНОМ ВРЕМЕНИ"

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101560558A (zh) * 2009-03-04 2009-10-21 中国检验检疫科学研究院 多重pcr产物的悬浮芯片检测方法
CN102943128A (zh) * 2012-10-29 2013-02-27 中国检验检疫科学研究院 一种针对蚊媒病毒的悬浮芯片多重非诊断性检测方法
CN103173567A (zh) * 2013-03-27 2013-06-26 中国检验检疫科学研究院 一种针对蜱传病原的悬浮芯片多重非诊断性检测方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
WINSLOW ET AL.: "Antibody-Mediated Elimination of the Obligate Intracellular Bacterial Pathogen Ehrlichia chaffeensis during Active Infection", INFECTION AND IMMUNITY, vol. 68, no. 4, April 2000 (2000-04-01), pages 2187 - 2195 *

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