201000631 九、發明說明： 【發明所屬之技術領域】 本發明係關於一種病毒之檢測方法，特別是關於一 種結合nested PCR及磁珠核酸探針雜合技術之家禽里奥 病毒檢測方法^ 【先前技術】201000631 IX. Description of the Invention: [Technical Field] The present invention relates to a method for detecting a virus, and more particularly to a method for detecting a poultry virus in combination with a nested PCR and a magnetic hybridization technique of a magnetic bead nucleic acid probe. 】
家禽里奥病毒(avian reovirus, ARV)，係一種會感染 禽鳥類之病毒，其基因體係由10條雙股RNA (dsRNA) 所構成’依分子量大小可分成L、Μ及S等基因群，至少 可轉澤出10種結構蛋白（λΑ、λΒ、A_C、//A、//Β、 私BC、//BN、crC、σ A及σΒ)以及4種非結構蛋白（ #NS、cjNS、Ρ10及Ρ17)。其中，S基因群包含SI、S2 、S3及S4等四段雙股RNA; S1基因所轉譯的蛋白 ，該JC蛋白可誘發禽烏類產生型專一性中和抗體；群專 一性抗體則由S3基因轉譯的σΒ蛋白所誘發產生。病毒 ^ S2基因轉譯之蛋白為σΑ，已被證實具有結合雙股RNA 的月b力，亦可能與病毒的干擾素抗性有關。在不同血清型 、致病型及基因型的家禽里奥病毒中，σΑ蛋白之基因序 . 列具有高度保留性；而S4基因轉譯之aNS蛋白則具單股 * RNA之結合能力。受家禽里奥病毒所感染之禽鳥類，容 易引起腸道疾病、呼吸道疾病、心肌炎、肝炎、吸收不良 症候群及病毋性關節炎等病症；雖然感染家禽里奥病毒之 禽鳥類死亡率不高，但由於該等病症會造成禽鳥類跋腳或 活動力減低等症狀，進而使禽鳥類之進食能力及換肉率下 201000631 降，造成該禽鳥類之飼養者經濟損失。而研發可早期檢測 禽鳥類是否感染家禽里奥病毒之檢測方法係為不可忽視之 重要課題，以進一步進行受感染的禽鳥類之隔離，進而預 防里奥病毒之持續擴散感染，使飼養者之經濟損失降到最 低0 家禽里奥病毒傳統之診斷方式，係利用禽鳥類的抗 體力價之高低作為判定禽鳥類是否遭受感染的依據；但此 法受限於檢體收集所需時間長，而無法達到早期偵測之效 果。近年來，由於分子生物技術的發展，利用核酸技術診 斷疾病的方式逐漸取代傳統的疾病診斷方式，核酸技術之 診斷方式可在禽鳥類之抗體力價尚未上升的空窗期即可偵 測病毒核酸，以確認該禽鳥類是否遭受病毒感染。然而， 目前家禽里奥病毒之分子診斷技術包括反轉錄聚合？連鎖 反應(reverse transcription polymerase chain, RT-PCR)、real time RT-PCR 或 DNA 晶片技術（DNA microarray)。然而 因為RT-PCR之分析靈敏度及特異性低，在禽鳥類受病毒 感染之初期’該病毒量尚低，以RT-PCR技術進行檢測容 易偵測不到病毒；而real-time RT-PCR雖然可定量樣本且 分析時間快，但易產生非特異性反應；另外，DN A晶片 技術需將病原之cDNA固定於晶片上，而使樣本與 晶片的雜合反應效率變差，雜合時間需長達16小時；且 DNA晶片技術需卬貝的儀器設備，如點片機或訊號掃描 儀等’其造成欲檢測病毒者之成本負擔。 在2004年’ Wang等學者以磁珠核酸探針系統偵測 201000631 -、结核桿g，此技術可大幅提升病毒侧之靈敏度及特異性 ，5以呼吸道檢體為樣本時，其靈敏度及特異性分別為 66.7%與97.2%。另外，以磁珠核酸探針系統檢測抹片陴 性樣本，丨靈敏度及特異性皆高於傳、統之BD Pr〇beT: ET Direct TB System (DTB)及傳統的抹片檢測方法。另外 ，2005年亦有磁珠核酸探針系統檢測牛流行熱病毒之報 σ’χ表，作者比較傳統RT PCR、reai_time rt_pcr及磁 ， 珠核酸探針系統之靈敏度，分㈣18.18%、36.36%及 72.73%。證實磁珠猶探針祕具高度錄性，適合低病 原量樣本之檢測。 【發明内容】 =刖尚無利用磁珠核酸探針系統偵測家禽里奥病毒 f研究發表。因此’本發日狀為改善傳㈣測家禽里奥病 毋方法之無法於病毒感染初期偵測、低靈敏度、低特異性 耗時且尚成本之缺點，進行努力研究而研發出一種可應 ί 祕制各種血清奴基目型之家輕奥病毒的磁 珠核酸 探針方法。 本發月之主要目的係提供一種家禽里奥病毒之檢測 • 方j，其係利用一磁珠核酸探針雜合技術，以檢測出相當 微里之病毒’使得本發明具有可提高檢測靈敏度之功效。 、本發明之次要目的係提供一種家禽里奥病毒之檢測 方=，其於RT-PCR步驟後，接續nested PCR之步驟， 係:別以外、内二組PCR引子對進行家禽里奥病毒基因 進行兩-人PCR擴增反應，崎低僅用RT_pCR擴增時所 —7 一 201000631 功效。陵反應’使得本發明具有提高檢挪特異性之 方法核目酸=提供—種家禽里細毒之檢測 僅以冷光儀侦測"技術係以冷光為訊號，而 。 使件本發明可減少债測所需之成本 酸探針，該磁珠磁珠結f成-磁珠核 因而提高探針的雜合效率，、/ 且可懸序於液體’ 間大幅縮短，使得^明且而使磁珠核酸探針雜合的時 效。 u具有可於短時間内完成檢測之功 引子=發Γίί龠一里目Γ系提供—種家禽里奥病毒檢測用 力千、，崎讀里奥病隸目 功:得本發明具有可大幅降低產生非特異性DN= 方法本目的係提供—種家禽里奥病毒之檢測 禽鳥感染家禽里奥病毒之初期，檢 存在，以進行禽鳥之隔離，避免家禽里奧病毒之 感木持、，概，㈣贿低家禽鱗者成本之損失。 本發㈣提供-縣禽里奥錢之檢财法，盆係 辦賊騎套組（咖_ ea ase A Probing kit)，該磁珠核酸探針套咭 合⑽及磁__針雜合技術，以大幅提昇偵 201000631 測家纽奥病毒枝之錄度及特異性。 【實施方式】 為了讓本發明之上述和其他目的、特徵和優點能更 明確被了解’下文將特舉本發明較佳實施例，並配合所附 圖式，作詳細說明如下。 请參照第1圖所示，本發明家禽里奥病毒之檢測方 法之較佳實施例係先以一 RT_PCR步驟對待測物基因作第 一次家禽里奥病毒基因增值，再取該RT_PCR之產物進行 一 nested PCR之步驟對該待測物基因作第二次之家禽里 奥病毒基因之增值，以降低僅用RT_pCR擴增家禽里奥病 毒基因時所產生的非特異性反應；接續取該nested pcR 之產物進行一磁珠核酸探針雜合反應及訊號偵測，以提高 檢測家禽里奥病毒基因之靈敏度及大幅縮短檢測所花費之 時間。The avian reovirus (ARV) is a virus that infects birds. Its genetic system consists of 10 double-stranded RNAs (dsRNAs). According to the molecular weight, it can be divided into L, Μ and S gene groups. It can transfer 10 structural proteins (λΑ, λΒ, A_C, //A, //Β, BC, //BN, crC, σ A and σΒ) and 4 non-structural proteins (#NS, cjNS, Ρ10) And Ρ17). Among them, the S gene group contains four pairs of double-stranded RNAs such as SI, S2, S3 and S4; the protein translated by the S1 gene, the JC protein can induce avian-specific neutralizing antibody; the group-specific antibody is composed of S3 Gene-translated σΒ protein is induced. The virus ^ S2 gene translated protein is σΑ, which has been shown to have a monthly b-binding force in binding to double-stranded RNA, and may also be associated with interferon resistance of the virus. Among the different serotypes, pathogenic types and genotypes of the poultry virus, the σΑ protein gene sequence is highly retained; while the S4 gene translated aNS protein has a single strand * RNA binding ability. Birds infected with the poultry Rio virus are prone to intestinal diseases, respiratory diseases, myocarditis, hepatitis, malabsorption syndrome and disease-induced arthritis; although the mortality rate of birds infected with the Leo virus is not high, However, due to the fact that these diseases may cause symptoms such as lameness or reduced mobility of birds, the bird's feeding ability and meat exchange rate will fall below 201,000631, resulting in economic loss of the bird's breeder. The development of an early detection method for the detection of whether poultry birds are infected with poultry virus is an important issue that cannot be ignored to further isolate the infected birds, thereby preventing the continued spread of the Rio virus and the economy of the breeder. The loss is reduced to a minimum of 0. The traditional diagnosis method of the Avian virus is based on the use of the antibody price of the bird as the basis for determining whether the bird is infected. However, this method is limited by the long time required for the collection of the sample. Achieve the effect of early detection. In recent years, due to the development of molecular biotechnology, the use of nucleic acid technology to diagnose diseases has gradually replaced the traditional method of disease diagnosis. The diagnostic method of nucleic acid technology can detect viral nucleic acid in the window of the bird's antibody price. To confirm whether the bird is infected with a virus. However, the current molecular diagnostic techniques for poultry virus include reverse transcription polymerization. Reverse transcription polymerase chain (RT-PCR), real time RT-PCR or DNA microarray. However, because of the low sensitivity and specificity of RT-PCR analysis, the amount of the virus is still low in the early stage of virus infection of birds. It is easy to detect the virus by RT-PCR, while real-time RT-PCR is The sample can be quantified and the analysis time is fast, but it is easy to produce non-specific reaction. In addition, the DN A wafer technology needs to fix the cDNA of the pathogen on the wafer, and the hybridization efficiency of the sample and the wafer is deteriorated, and the hybridization time needs to be long. Up to 16 hours; and DNA wafer technology requires mussel instruments, such as spot machines or signal scanners, which cause the cost of the virus to be detected. In 2004, Wang and other scholars used the magnetic bead nucleic acid probe system to detect 201000631 - and tuberculosis rod g. This technique can greatly improve the sensitivity and specificity of the virus side. 5 When the respiratory sample is used as a sample, its sensitivity and specificity. They were 66.7% and 97.2% respectively. In addition, using the magnetic bead nucleic acid probe system to detect smear samples, the sensitivity and specificity of sputum are higher than those of BD Pr〇beT: ET Direct TB System (DTB) and traditional smear detection methods. In addition, in 2005, the magnetic bead nucleic acid probe system was also used to detect the report of bovine epidemic fever virus. The author compared the sensitivity of traditional RT PCR, reai_time rt_pcr and magnetic, bead nucleic acid probe system, and divided (4) 18.18%, 36.36 % and 72.73%. It is confirmed that the magnetic beads are highly visible to the probe and are suitable for the detection of low pathogenic samples. [Summary of the Invention] = The use of the magnetic bead nucleic acid probe system to detect poultry virus virus f has not been published. Therefore, the 'this hair shape is a method for improving the transmission of the disease. The method of measuring the disease of the poultry is not able to detect the initial stage of virus infection, low sensitivity, low specificity, time and cost, and research and develop an effective solution. A magnetic bead nucleic acid probe method for secreting a variety of serum nucleus type homes. The main purpose of this month is to provide a test for poultry virus, which uses a magnetic hybridization technique to detect a relatively small virus. This makes the invention more sensitive to detection. efficacy. The secondary object of the present invention is to provide a test for a poultry virus, which is followed by a step of nested PCR after the RT-PCR step, and the two pairs of PCR primer pairs are used to carry out the poultry virus gene. A two-human PCR amplification reaction was performed, and the effect was only 7-81000631 when the RT_pCR was amplified. The linguistic reaction makes the invention have the method of improving the specificity of detection. Nucleic acid=providing the detection of the fine poison in the poultry is only detected by the luminometer, and the technology is based on the cold light. The present invention can reduce the cost of the acid probe required for the debt measurement, and the magnetic bead of the magnetic bead becomes a magnetic bead core, thereby improving the hybrid efficiency of the probe, and / can be greatly shortened between the liquids. The aging effect of making the magnetic bead nucleic acid probe heterozygous. u has the ability to complete the test in a short time. 引 Γ 龠 ί ί 龠 里 提供 提供 提供 — 种 家 家 家 家 家 家 家 家 家 家 家 家 家 家 家 家 家 家 家 家 里奥 里奥 里奥 里奥 里奥 里奥 里奥 里奥 里奥 里奥 里奥 里奥 里奥Non-specific DN= Method The purpose of this study is to provide a test for the detection of poultry birds infected with poultry, and to detect the isolation of birds and birds, and to avoid the feeling of poultry and virus. (4) The loss of the cost of bribery of poultry scales. This issue (4) provides the method of checking the county poultry rio rio money, the pot es ea ase A Probing kit, the magnetic bead nucleic acid probe set (10) and the magnetic __ needle hybrid technology In order to greatly enhance the record and specificity of the detective New York virus. The above and other objects, features, and advantages of the present invention will become more apparent <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; Referring to FIG. 1 , a preferred embodiment of the method for detecting a poultry virus of the present invention is to first use the RT_PCR step to perform the first poultry virus gene gene addition, and then take the product of the RT_PCR. A nested PCR step is used to increase the value of the poultry virus gene for the second time to reduce the non-specific reaction generated by amplifying the poultry virus gene only by RT_pCR; and subsequently taking the nested pcR The product is subjected to a magnetic bead nucleic acid probe heterozygous reaction and signal detection to improve the sensitivity of detecting the poultry virus gene and greatly shorten the time taken for the detection.
RT-PCR 本發明先依據家禽里奥病毒疫苗株S1133之S2基因 ^ 所轉譯的高度保留序列設計一特異性最佳之引子對，以作 為RT-PCR增幅crA蛋白基因之用，該引子對係為S2F3 :5’ -GGCTTCTACTTCTCCTCGAAGACTC-3，與 S2R3 :5 ’ -AGAAGTCATTAGCCTCCTGCGTTA-3 ’ （產物大 小為 388 bp )。 本發明為確定該S2F3/S2R3引子對僅對家禽里奥病 毒具有特異性，取禽類相關病毒、非禽類相關病毒或其他 動物病毒之DNA測試RT-PCR引子是否產生非特異性之 201000631 极Γ該禽類病毒係可選擇採用禽流感病毒、傳染 乳官火病毒、傳染性華氏囊病病毒或新城病病毒；直 毒ί可選擇獅如牛流行熱病毒、犬_病毒或 減測試是否產生非特異性擴增之產物；請參RT-PCR The present invention first designs a specific primer pair based on the highly retained sequence translated by the S2 gene of the poultry virus vaccine strain S1133, and uses it as an RT-PCR amplification crA protein gene. S2F3: 5'-GGCTTCTACTTCTCCTCGAAGACTC-3, and S2R3: 5'-AGAAGTCATTAGCCTCCTGCGTTA-3' (product size is 388 bp). The present invention is to determine whether the S2F3/S2R3 primer pair is specific only to the poultry virus, and whether the DNA test RT-PCR primer for avian-associated virus, non-avian-associated virus or other animal virus produces a non-specific 201000631 limit Avian viruses can choose to use avian influenza virus, infectious milk fire virus, infectious bursal disease virus or Newcastle disease virus; direct poisoning can choose lions such as cattle epidemic fever virus, canine virus or reduction test to produce non-specific Product of amplification; please refer to
<、Α 及 2C 圖所示’其中 Marker 為 Bio-IGOTM DNA AIV為禽流感病毒，卿v為牛流行熱病毒， CDV為H熱病毒，CSFV為_病毒，my為傳染性支 氣管炎病毒’ IBDV為傳染性華氏囊病病毒，NDV為新城 ，病毒’ NTC為陰性對照虹，ARV為家禽里奥病毒；如 弟2C圖所示’首先以各禽類病毒及其他動物病毒之特定 引子對各禽類病毒及其他動物病毒之dna騎RT_pcR 反應以確&忍各禽類病毒及其他動物病毒·A之可用性 ，結果顯示各有其特異性產物被擴增，證明各禽類病毒及 其他動物病毒之DNA皆為可用之樣本。如第2A圖所示 ’結果可1 S2F3/S2R3弓丨子對之特異性佳，對禽類病毒<, Α and 2C are shown in 'Where Marker is Bio-IGOTM DNA AIV is avian influenza virus, Qing v is cattle fever virus, CDV is H heat virus, CSFV is _ virus, and my is infectious bronchitis virus' IBDV is an infectious bursal disease virus, NDV is a new city, virus 'NTC is a negative control rainbow, ARV is a poultry virus; as shown in Figure 2C', first of all the specific primers for each avian virus and other animal viruses The virus and other animal viruses dna ride the RT_pcR reaction to confirm the availability of the various avian viruses and other animal viruses A. The results showed that each specific product was amplified, and the DNA of each avian virus and other animal viruses was confirmed. A sample that is available. As shown in Figure 2A, the results are as good as 1 S2F3/S2R3, which is good for avian viruses.
及其他動物鱗錢增反應，歸家禽里奥病毒反應出特 異性產物。And other animal scales increase the reaction, return to the poultry virus to reflect the specific product.
在確認S2F3/S2R3引子對僅對家禽里奥病毒具有特 異性後，本發明於實際操作RT_PCR反應時，係先將待測 物之基因反轉錄成cDNA，取1至5 μΐ之該待測物cDNA 與PCR緩衝液、dNTPs、S2R3/S2F3引子對及DNA聚 合？均勻混合，並置於90至95。(：作用約30秒至5分鐘 ’使cDNA模板完全變性；接著於37至7〇 〇c下作用約 30秒至3分鐘’較佳係選用62。(：為RT-PCR之煉合溫度 201000631 ，以使該待測物之cDNA與S2R3/S2F3弓丨子煉合；再於 65至80它下作用約3〇秒至5分鐘，以使dna聚合酶 進行DNA之合成；以上之DNA擴增步驟較佳係進行°2〇 至40個循環；最後，可在DNA聚合？進行dna之合成 的階段延長其作用時間，以增加擴增效率；取部分第一次 PCR之產物以電泳分析及溴化乙錠染色，並以紫外燈照 射加以鑑定該產物之大小是否為388 bp，以確定該產物之 準確性。After confirming that the S2F3/S2R3 primer pair is specific only to the poultry virus, the present invention reversely transcribes the gene of the analyte into cDNA after taking the RT_PCR reaction, and takes 1 to 5 μM of the analyte. cDNA and PCR buffer, dNTPs, S2R3/S2F3 primer pair and DNA polymerization? Mix evenly and place at 90 to 95. (: the effect is about 30 seconds to 5 minutes 'to completely denature the cDNA template; then about 37 to 7 〇〇c for about 30 seconds to 3 minutes'. The preferred system is 62. (: RT-PCR refining temperature 201000631 So that the cDNA of the test substance is smelted with S2R3/S2F3 scorpion; and then it is subjected to 65 to 80 for about 3 sec to 5 minutes to allow DNA polymerase to synthesize DNA; the above DNA amplification The step is preferably carried out from 2 to 40 cycles; finally, the action time can be extended in the stage of DNA polymerization and DNA synthesis to increase the amplification efficiency; and the products of the first PCR are analyzed by electrophoresis and bromine. Ethigen was stained and the size of the product was identified as 388 bp by irradiation with a UV lamp to determine the accuracy of the product.
Nested PCR 本發明先依據家禽里奥病毒疫苗株S1133之S2基因 所轉譯的高度保留序列設計一特異性最佳，且其增幅大小 小於第一次PCR反應之產物的引子對，以作為Nested PCR增幅σΑ蛋白基因之用，該引子對係為S2F5 : 5，_ ACTTAACCGCTATCGTCATGGCTTG-3，與 S2R5 : 5，- CCTGCGCGGTAGTCAGTAAGTTGAG-3 ’ 之引子對（產 物大小為144 bp) ’為確定該S2F5/S2R5引子對僅對家禽 里奥病毒具有特異性，本發明另取於RT-PCR步驟中所述 之各種病毒測試nested PCR引子是否產生非特異性之反 應。請參照第2B圖所示，其中Marker為Bi〇-1〇〇TM DNA Ladder· ’ AIV為禽流感病毒，BEFV為牛流行熱病毒 ’ CDV為犬瘟熱病毒，CSFV為豬瘟病毒，IBV為傳染性 支氣管炎病毒，IBDV為傳染性華氏囊病病毒’ NDV為新 城病病毒’ NTC為陰性對照組，ARV為家禽里奥病毒； 結果顯示’在禽類病毒及其他動物病毒亦同樣無產物被擴 201000631 增’而在豕禽里奥病毒則有特異性產物被增幅（144 bp)。 . 在確認S2F5/S2R5引子對僅對家禽里奥病毒具有特 異性後，本發明於實際操作nested PCR反應時，先取上 述之RT-PCR產物DNA與PCR緩衝液、dNTPs、標誌有 生物素之S2R5/S2F5引子對及DNA聚合？均勻混合，再 置於90至95 C作用約30秒至5分鐘，使家禽里奥病毒 第一次PCR產物DNA模板完全變性；接著於37至7〇 , °C下作用約30秒至3分鐘，較佳係選用55。(：為nested PCR之煉合溫度’以使家禽里奥病毒第一次PCR產物 DNA與S2R5/S2F5引子煉合；再於65至80。(：下作用约 30秒至5分鐘，以使DNA聚合？進行DNA之合成；以 上之DNA擴增步驟較佳係進行20至40個循環；最後， 可在DNA聚合？進行DNA之合成的階段延長其作用時 間，以增加家禽里奥病毒DNA之擴增效率；取部分 nested PCR產物以電泳分析後，以溴化乙錠染色，並以紫 外燈照射加以鑑定該產物之大小是否為144 bp，以確定該 %、-- 產物之準確性。 磁珠核酸探針雜合反應 .本發明家禽里奥病毒核酸探針乃根據12株不同血清 型及基因型之家禽里奥病毒S2基因序列而設計，選取核 酸序列較保留之區域以設計具特異性的核酸探針(probe 1) :5，-GD*CCTGGCTTTTCCATGAATGG-3，（D*= A，T，G) ’或以與該核酸探針鹼基序列互補之鹼基序列設計具特異 性的核酸探針。再以magnetic bead coupling kit將具特異 ——12—— 201000631 性的核酸探針與磁珠騎結合，时絲禽里奥病毒磁珠 核s夂探針，此合成豕禽里奥病毒磁珠核酸探針之反應係委 託亞洲基因科技股份有限公司（AsiaGen⑶π〇^ί〇η， Tainan，Taiwan)所完成。 而磁珠探針系統之詳細操作流程如下，先取上述 nested PCR之雙股DNA產物’可將其稀釋5〇至2⑻倍， 本發明較佳係將該nested PCR之雙股〇财產物稀釋1〇〇 倍，再與去離子水、雜合緩衝液(5X ssc，〇 5% SDS)及磁 珠核酸楝針均勻^合於—雜合反應管内，其中該磁珠核酸 探針之濃度可為10 #M、12 #M、14 及16 ，本發明較佳選用10#M之磁珠核酸探針濃度；再將該 雜合反應管置於95°c，作用5至10分鐘，使雙股的 DNA模板完全變性成單股〇να ;再於50至60。(：之雜合 溫度下作用10至20分鐘，以進行磁珠核酸探針與該單股 DNA雜合反應’其中該雜合溫度較佳可選用51<^。接著 將雜合反應管移至磁性試管架上3至5分鐘，以分離液體 與該經過雜合反應之磁珠，並將液體吸除；接著，加入預 熱好之洗滌緩衝液於該雜合反應管内並震盪混合數秒後， 置回磁性試管架3至5分鐘，以分離液體與該經過雜合反 應之磁珠；接著再將液體吸除，以洗滌該經過雜合反應之 磁珠’此步驟可洗條數次。再加入阻滯缓衝液（bl0cking solution，含駱蛋白）與生物活性？之混合液於該雜合反 應管内’以與該洗滌完善之經過雜合反應的磁珠充分震盡 混合’再將雜合反應管置回磁性試管架3至5分鐘，並吸 —13 — 201000631 除液體。隨後加入5〇〇 //1之含0·5% Tween 20的磷酸緩 衝液（PBST)於該雜合反應管内，並震盪加以洗滌數秒 ’再置回磁性試管架3至5分鐘並將液體吸除，此步驟可 洗滌數次。最後’加入磷酸緩衝液（1 χ pBS)於該雜合 反應官内’並充分震盪，以將該雜合產物由該磁珠上洗下 ’使該雜合產物懸浮於液體。取該含有雜合產物之液體與 新鮮備製之冷光受質混合液充分混合，並在冷光儀下偵測 冷光值。 核酸磁珠探針套組之靈敏度分析 取豕禽里奥病毒S1133毒珠之基因體當作模板，以 分光光度計測得核酸吸光值（〇D26G)，並計算出核酸濃度 (g/L) ’連續稀釋病毒核酸為1〇5、1〇4、1〇3、102、10、5 及1拷貝數///1，並各取1 病毒核酸當作模板，作 RT-PCR、nested-PCR及磁珠核酸探針雜合反應，結果顯 示於弟3A至3C圖。如第3A圖所示’其中；結果顯示以 105、104、103、102、1〇、5及1拷貝數/ 的家禽里 奥病毒S1133毒株之基因體存在時，磁珠核酸探針套組所 偵測之冷光比值分別為26,538、23,434、21，658、15,465 、10,731、2,283及66 ’而陽性對照為家禽里奥病毒 S1133毒株’其冷光比值為34,693 ;陰性對照之冷光比值 則為61。由此可見，磁珠核酸探針套組最低可偵測約5 拷貝數/ //1之家禽里奥病毒S1133毒株之基因體。第3B 及3C圖則分別為不同拷貝數的家禽里奥病毒S1133毒株 之RT-PCR及nested PCR結果，其分別可偵測約ι〇4拷 201000631 貝數/ //1及ίο拷貝數/ "之家禽里奥病毒sll33毒株之 f因體’顯而易見的’本發明增加nested PCR與磁珠核 酉欠探針檢測的步驟，相較於僅用rt_pcr進行檢測，本發 月可偵測低之家禽里奥病毒量，而可提高了檢測之靈敏 度。 核酸磁珠探針套組之特異性分析 主以核酸磁珠探針系統分析禽流感病毒、牛流行熱病 毒、犬瘟熱病毒、豬瘟病毒、傳染性支氣管炎病毒、傳染 眭華氏囊病病毒及新城病病毒，而家禽里奥病毒則為陽性 對照’於冷光儀分析個別之冷光值，以檢測套組之特異性 °如第4 _示’其中Y軸為樣本與陰性對照組之冷光Nested PCR The present invention firstly designs a primer having the best specificity based on the highly retained sequence translated by the S2 gene of the poultry virus vaccine strain S1133, and the amplification amount is smaller than that of the product of the first PCR reaction, as a Nested PCR increase. For the σΑ protein gene, the primer pair is S2F5: 5, _ ACTTAACCGCTATCGTCATGGCTTG-3, and the pair of S2R5: 5,- CCTGCGCGGTAGTCAGTAAGTTGAG-3 ' (product size is 144 bp) 'to determine the S2F5/S2R5 primer pair only Specific to the poultry rio virus, the present invention further measures whether the nested PCR primers of the various viruses described in the RT-PCR step produce a non-specific reaction. Please refer to Figure 2B, where Marker is Bi〇-1〇〇TM DNA Ladder· 'AIV is avian influenza virus, BEFV is bovine epidemic fever virus' CDV is canine distemper virus, CSFV is swine fever virus, IBV is Infectious bronchitis virus, IBDV is infectious bursal disease virus 'NDV is Newcastle disease virus' NTC is a negative control group, ARV is a poultry virus; the results show that 'the avian virus and other animal viruses are also no product to be expanded 201000631 increased 'and the specific product was increased (144 bp) in the cockroach. After confirming that the S2F5/S2R5 primer pair is specific only to the poultry virus, the present invention first takes the above RT-PCR product DNA and PCR buffer, dNTPs, and biotinylated S2R5 in the actual operation of the nested PCR reaction. /S2F5 primer pair and DNA aggregation? Mix evenly, then place at 90 to 95 C for about 30 seconds to 5 minutes to completely denature the DNA template of the first PCR product of the poultry virus, and then apply it for about 30 seconds to 3 minutes at 37 to 7 °C. Preferably, 55 is used. (: is the melting temperature of nested PCR 'to make the first PCR product DNA of the poultry virus to be combined with the S2R5/S2F5 primer; and then 65 to 80. (: the next action is about 30 seconds to 5 minutes to make the DNA Polymerization? Perform DNA synthesis; the above DNA amplification step is preferably carried out for 20 to 40 cycles; finally, the action time can be prolonged in the stage of DNA polymerization and DNA synthesis to increase the expansion of the poultry virus DNA. Increasing efficiency; taking some of the nested PCR products for electrophoresis analysis, staining with ethidium bromide, and identifying whether the size of the product is 144 bp by ultraviolet lamp irradiation, to determine the accuracy of the %, --- product. Hybridization reaction of nucleic acid probe. The poultry virus virus nucleic acid probe of the present invention is designed according to 12 different serotypes and genotypes of the poultry virus S2 gene sequence, and the nucleic acid sequence is selected to be more specific to design specificity. Nucleic acid probe (probe 1): 5, -GD*CCTGGCTTTTCCATGAATGG-3, (D*= A, T, G) ' or design a specific nucleic acid probe with a base sequence complementary to the base sequence of the nucleic acid probe Needle. Then use the magnetic bead coupling kit to —————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————— Technology Co., Ltd. (AsiaGen (3) π〇^ί〇η, Tainan, Taiwan) completed the detailed operation of the magnetic bead probe system as follows, first take the above-mentioned nested PCR double-strand DNA product 'can be diluted 5 to 2 (8) times Preferably, in the present invention, the nested PCR double-stranded property is diluted 1 time, and then uniformly mixed with deionized water, hybrid buffer (5X ssc, 〇5% SDS) and magnetic beads nucleic acid needle. In the heterozygous reaction tube, wherein the concentration of the magnetic bead nucleic acid probe can be 10 #M, 12 #M, 14 and 16, and the concentration of the magnetic bead nucleic acid probe of 10#M is preferably used in the present invention; The reaction tube is placed at 95 ° C for 5 to 10 minutes to completely denature the double-stranded DNA template into a single strand of 〇να; and then at 50 to 60. (: at a heterozygous temperature for 10 to 20 minutes, for The magnetic bead nucleic acid probe is hybridized with the single-strand DNA, wherein the hybridization temperature is preferably 51 <^. The hybrid reaction tube is moved to the magnetic tube rack for 3 to 5 minutes to separate the liquid from the hybridized magnetic beads, and the liquid is aspirated; then, the preheated washing buffer is added to the hybrid reaction. After mixing and shaking for several seconds in the tube, the magnetic tube rack is returned to the magnetic tube rack for 3 to 5 minutes to separate the liquid and the hybridized magnetic beads; then the liquid is aspirated to wash the hybrid magnetic beads. Wash the strip several times. Adding blocking buffer (bl0cking solution, containing lignin) and biological activity? The mixture is mixed in the hybrid reaction tube with the magnetic beads that have undergone the hybridization reaction, and then the hybrid reaction tube is returned to the magnetic tube rack for 3 to 5 minutes, and sucked - 13 - 201000631 In addition to liquids. Then add 5 〇〇//1 of 0.5% Tween 20 phosphate buffer (PBST) in the hybrid reaction tube, and shake for a few seconds to re-set the magnetic tube holder for 3 to 5 minutes and suck the liquid. In addition, this step can be washed several times. Finally, a phosphate buffer (1 χ pBS) is added to the heterogeneous reaction unit and shaken sufficiently to wash the hybrid product from the magnetic beads to suspend the hybrid product in a liquid. The liquid containing the hybrid product is thoroughly mixed with the freshly prepared cold light receiving mixture, and the cold light value is detected under a cold light meter. Sensitivity analysis of the nucleic acid magnetic bead probe set The genomic volume of the venom virus S1133 venom was used as a template, and the absorbance of the nucleic acid (〇D26G) was measured by a spectrophotometer, and the nucleic acid concentration (g/L) was calculated. Serial dilution of viral nucleic acids is 1〇5, 1〇4, 1〇3, 102, 10, 5 and 1 copy number///1, and each adopts 1 viral nucleic acid as a template for RT-PCR, nested-PCR and The magnetic bead nucleic acid probe was heterozygous and the results were shown in the 3A to 3C map. As shown in Figure 3A, the results show that the magnetic bead nucleic acid probe set exists in the presence of 105, 104, 103, 102, 1〇, 5, and 1 copy number of the genome of the poultry virus S1133 strain. The detected cold light ratios were 26,538, 23,434, 21,658, 15,465, 10,731, 2,283 and 66 ', respectively, while the positive control was the poultry virus S1133 strain's cold-light ratio 34,693; the negative control's cold-light ratio was 61. . It can be seen that the magnetic bead nucleic acid probe set can detect a genome of about 5 copies/ //1 of the poultry virus S1133 strain. Figures 3B and 3C are RT-PCR and nested PCR results of different copy number of poultry virus S1133 strains, respectively, which can detect about ι〇4 copy 201000631 number of shells / /1 and ίο copy number / "The poultry virus of the poultry virus sll33 strain 'obvious' is obvious. The present invention increases the steps of nested PCR and magnetic bead nucleus under probe detection, compared with the detection using only rt_pcr, this month can detect The low amount of poultry virus can increase the sensitivity of detection. Specific analysis of nucleic acid magnetic bead probe kits. Analysis of avian influenza virus, bovine epidemic fever virus, canine distemper virus, swine fever virus, infectious bronchitis virus, infectious sputum bursal disease virus by nucleic acid magnetic bead probe system And the Newcastle disease virus, while the poultry virus is a positive control 'analyze the individual cold light values in the cold light meter to check the specificity of the set. For example, the 4th_show' where the Y axis is the cold light of the sample and the negative control group
比值’ x轴代表各種禽類相關病毒及非相關病毒，NTC ^陰性對n AIV為纽感鱗，BEFV為牛流行熱病 毒，0)V為犬瘟熱病毒，cSFV為豬瘟病毒，IBV為傳染 性支氣管炎病毒，IBDV為傳染性華氏囊病病毒，^^^乂為 ，城病病毒’ ARV為陽性對照組（家禽里奥病毒S1133 母株）。樣本與陰性對照比較之P值小於0.05 ;結果顯示 $机感病毒、牛流行熱病毒、犬瘟熱病毒、豬瘟病毒、傳 、木性支氣官炎病毒、傳染性華氏囊病病毒及新城病病毒之 ^光比值分別為 131、105、85、93、121、114 及 1〇2 ; 陽性對照組家禽里奥病毒S1133毒株之冷光比值為48,819 ’陰性對照之冷光比值則為115。顯而易見的，磁珠核酸 探針套組與禽類病毒及其他動物病毒無交叉反應，特異性 佳。 —15—— 201000631 核酸磁珠探針套組之再現性分析 再現性之優劣則可以變異係數(cv)作為參考之依據 ’變異係數為標準偏差(SD)相對於檢測平均值之百分比， 此數值無單位，且恆為正數。當檢測值不穩定時，標準偏 差相對增加，變異係數亦高，此則代表分析方法之再現性 差，精密度不良；而一般實驗室檢測方法可接受之變異係 數需小於10%，但當受測物質濃度極穩定時，變異係數則 需小於5%。本發明係取家禽里奥病毒SU33毒株作測試 樣本，在同一次核酸磁珠探針檢測之實驗内作三次重複（ Intra-assay)，並於同曰内分別獨立操作三回（Within day )’且連續測試三曰（Between-day)。結果如第5A圖所示 ，豕禽里奥病毒S1133毒株於同次間之冷光比值分別為 20,225、20,290及19,887 ; SD則為216 ;陰對照之冷光比 值為141、127及134 ; SD則為6.7。第5B圖顯示家禽里 奥病毒S1133毒株母日操作二回之冷光比值分別為19,〇84 、18,891 及 20，134 ; SD 則為 288、249 及 216 ;陰性對照 之冷光比值為125、110及99 ; SD則為5·4、3.2及4.4。 家禽里奥病毒S1133毒株連續操作三天之冷光比值分別為 20,106、19,370 及 20,320，SD 則為 80、668 及 241 ;陰性 對照之冷光比值為U8、130及111 ; SD則為1.5、9.4及 13.2。經由統計分析得知套組之同一實驗内作三次重複之 CV為1.1%，同日内分別獨立操作三回與連續操作三天 之CV分別為丨.3%及L7%，證實此套組之再現性佳，可 作一般常規檢驗之用。 201000631 測試臨床不同血清型及基因型之家禽里奥病毒 . 應用臨床上不同血清型及基因型之家禽里奥病毒樣 本測試本套組’以評估套組之臨床實用性。測試之病毒株 共分為三大群，其中S1133、1733及2408為疫苗株，R2/ TW、T6、601G、916、918、919、1017-1 及 750505 為台 灣野外分離株，OS161則為曰本株。如第6圖所示，其中 Y轴為樣本與陰性對照組之冷光比值，X軸代表不同毒株 之豕禽里奥病毒，NTC為陰性對照組，樣本與陰性對照 比較之p值小於〇.05 ;結果顯示，各病毒株之冷光比值， 其中 S1133、1733 及 2408 為 40,949、36,252 及 26,332 ; R2/TW、T6、601G、916、918、919、1017-1 及 750505 貝1J 分別為 26,942、34,156、27,838、23,636、32,504、 29,645、33,788 及 34,402 ;而 OS161 則為 31，883。結果顯 不，本發明之磁珠核酸探針套組可偵測不同血清型及基因 型之家禽里奥病毒。 ί 由上述實驗可驗證本發明對家禽里奥病毒之特異性 ，因此使用本發明對一待檢測物進行檢測時，首先進行 第步驟：對一待檢測物進行rT_pcr ;接著進行第二步 * 驟，對第一步驟之產物進行nested PCR ;接著進行第三步 • 冑’取第二步驟之產物進行磁珠核酸探針反應，亦即以上 述之磁珠核酸探針與該第二步驟之產物進行雜合 此’便可準確檢測出該待測樣品令是否含有家禽里奥病毒 雖然本發明已利肖上述較佳實施例揭示，然其並非 17 — 201000631 用以限定本發明，任何熟習此技藝者在不脫離本發明之精 和顧之内’㈣上述實施舰行各種更動與修改仍^ 本發撕保護之技術範4，因林發明之倾 附之申請專利範圍所界定者為準。 田 —18 一— 201000631 【圖式簡單說明】 第1圖：家禽里奥病毒檢測方法流程圖。 第2圖：RT-PCR及nested PCR引子特異性反應之測試 第3圖··以家禽里奥病毒SU33毒株之基因體檢測磁珠 核酸探針套組之靈敏度。 第4圖 第5圖 第6圖 毋'之結果。 磁珠核酸探針套組之特異性分析。 磁珠核酸探針套組之再現性分析。 病 測試臨床上不同血清型及基因型的家禽里奥 【主要元件符號說明】 201000631 序列表 <110>國立屏東科技大學 <120〉家禽里奥病毒之檢測方法 <130> <140> < 141 > <160> 5 <210> 1 <211> 25 <211 > DNA <213>人工序列 <220〉 <223> S2F3 <400> 1 ggcttctacttctcctcgaagactc <210> 2 <211> 24 <211> DNA <213>人工序列 <220> <223> S2R3 <400> 2 agaagtcattagcctcctgcgtta <210> 3 <211> 25 <211> DNA <213〉人工序列 <220> <223> S2F5 <400〉3 acttaaccgctatcgtcatggcttg —20 — 201000631 <210> 4 <211> 25 <211> DNA 、 <213>人工序列 <220> ^ <223〉S2R5 ‘ <400> 4 cctgcgcggtagtcagtaagttgag <210> 5 <211> 22 <211 > DNA <213>人工序列 <220> < 223 > probe 1 <400> 5 gd*cctggcttttccatgaatgg (d*= a，t，g) 一 21 —The ratio 'x' represents a variety of avian-associated and non-related viruses, NTC^negative vs. n AIV is New Zealand scale, BEFV is bovine epidemic fever virus, 0)V is canine distemper virus, cSFV is swine fever virus, IBV is infectious Bronchitis virus, IBDV is an infectious bursal disease virus, ^^^乂, and the urban disease virus 'ARV is a positive control group (poultry virus S1133 mother strain). The P value of the sample compared with the negative control was less than 0.05; the results showed that the machine virus, bovine epidemic fever virus, canine distemper virus, swine fever virus, transmission, woody bronchitis virus, infectious bursal disease virus and New City The light-to-light ratio of the disease virus was 131, 105, 85, 93, 121, 114 and 1〇2 respectively; the cold-light ratio of the positive control group of the poultry virus S1133 was 48,819 'the negative contrast ratio of the negative control was 115. Obviously, the magnetic bead nucleic acid probe set has no specific cross-reactivity with avian viruses and other animal viruses. —15 — 201000631 Reproducibility analysis of nucleic acid magnetic bead probe sets The probabilities of reproducibility can be based on the coefficient of variation (cv) as a reference. The coefficient of variation is the percentage of standard deviation (SD) relative to the mean value of the test. No unit, and always a positive number. When the detected value is unstable, the standard deviation is relatively increased, and the coefficient of variation is also high. This means that the reproducibility of the analytical method is poor and the precision is poor; while the variation coefficient acceptable for general laboratory testing methods is less than 10%, but when tested When the concentration of the substance is extremely stable, the coefficient of variation needs to be less than 5%. The invention adopts the poultry virus virus SU33 strain as a test sample, performs three repetitions (Intra-assay) in the same nucleic acid magnetic bead probe detection experiment, and independently operates three times in the same chamber (Within day). 'And continuously test Between-day. As shown in Fig. 5A, the ratio of luminescence ratio of S. sinensis strain S1133 strain was 20,225, 20,290 and 19,887 in the same period; SD was 216; the contrast ratio of yin control was 141, 127 and 134; SD Is 6.7. Figure 5B shows that the cold light ratio of the two-day operation of the poultry virus S1133 strain is 19, 〇84, 18,891 and 20,134; SD is 288, 249 and 216; the contrast ratio of the negative control is 125,110. And 99; SD is 5.4, 3.2 and 4.4. The luminescence ratio of the three-day continuous operation of the poultry virus S1133 strain was 20, 106, 19, 370 and 20,320, and the SD was 80, 668 and 241; the cold light ratio of the negative control was U8, 130 and 111; the SD was 1.5, 9.4 and 13.2. Through statistical analysis, it was found that the CV of the three replicates in the same experiment was 1.1%, and the CVs of three operations and three consecutive operations in the same day were respectively 丨.3% and L7%, confirming the reproduction of the group. Good quality, can be used for general routine inspection. 201000631 Testing of different serotypes and genotypes of poultry virus. The clinical trial of this kit was performed using a clinically different serotype and genotype of the poultry virus sample. The tested virus strains were divided into three groups, among which S1133, 1733 and 2408 were vaccine strains, R2/TW, T6, 601G, 916, 918, 919, 1017-1 and 750505 were Taiwan wild isolates, and OS161 was transcripts. Strain. As shown in Fig. 6, the Y-axis is the ratio of the luminescence ratio of the sample to the negative control group, the X-axis represents the scorpion virus of different strains, and the NTC is the negative control group. The p-value of the sample compared with the negative control is less than 〇. 05; The results show the luminescence ratio of each strain, of which S1133, 1733 and 2408 are 40,949, 36,252 and 26,332; R2/TW, T6, 601G, 916, 918, 919, 1017-1 and 750505 are 1st, respectively, 26,942, 34, 156, 27, 838, 23, 636, 32, 504, 29, 645, 33, 788 and 34, 402; and OS 161 is 31, 883. As a result, the magnetic bead nucleic acid probe set of the present invention can detect poultry virus of different serotypes and genotypes. ί The above experiment can verify the specificity of the present invention for the poultry virus, so when using the present invention to detect a sample to be detected, the first step is to perform rT_pcr on a sample to be detected; then proceed to the second step* Performing nested PCR on the product of the first step; then performing the third step of the second step of the product of the second step for the magnetic bead nucleic acid probe reaction, that is, the magnetic bead nucleic acid probe described above and the product of the second step It is possible to accurately detect whether the sample to be tested contains a poultry virus. Although the present invention has been disclosed in the above preferred embodiments, it is not 17-201000631 to define the present invention, and any skill in the art is known. Those who do not deviate from the essence of the present invention's (4) The above-mentioned implementation of the ship's various changes and modifications are still the technical norms of this tearing protection, which is subject to the definition of the patent application scope of the invention. Tian —18 一— 201000631 [Simple description of the diagram] Figure 1: Flow chart of the detection method of poultry virus. Figure 2: RT-PCR and nested PCR primer specific reaction test Figure 3 · Detection of magnetic beads probe set sensitivity with the genome of the poultry virus SU33 strain. Figure 4 Figure 5 Figure 6 The result of 毋 '. Specific analysis of magnetic bead nucleic acid probe sets. Reproducibility analysis of magnetic bead nucleic acid probe sets. Disease test clinically different serotypes and genotypes of poultry Rio [main component symbol description] 201000631 Sequence Listing <110> National Pingtung University of Science &Technology<120> Detection method of poultry virus] <130><140>< 141 ><160> 5 <210> 1 <211> 25 <211 > DNA <213> Artificial Sequence <220 <223> S2F3 <400> 1 ggcttctacttctcctcgaagactc <210> 2 <211> 24 <211> DNA <213>Artificial sequence<220><223> S2R3 <400> 2 agaagtcattagcctcctgcgtta <210> 3 <211> 25 <211> DNA <;213>Artifical sequence <220><223> S2F5 <400>3 acttaaccgctatcgtcatggcttg — 20 — 201000631 <210> 4 <211> 25 <211> DNA, <213> artificial sequence <220> ^ <223>S2R5 ' <400> 4 cctgcgcggtagtcagtaagttgag <210> 5 <211> 22 <211 > DNA <213> Artificial Sequence <220>< 223 > probe 1 <400> 5 gd*cctggcttttccatgaatgg (d*= a,t,g) A 21 —