TW200927939A

TW200927939A - Specific primers, oligo-nucleotide probes, biochip of dengue viruses identification, and identifying method thereof

Info

Publication number: TW200927939A
Application number: TW096150837A
Authority: TW
Inventors: Wu-Chun Tu; Kuang-Hui Lu; Ming-Cheng Wu
Original assignee: Univ Nat Chunghsing
Priority date: 2007-12-28
Filing date: 2007-12-28
Publication date: 2009-07-01
Also published as: TWI381053B

Abstract

Specific primers, oligo-nucleotide probes, a biochip of dengue viruses identification, and the identifying method thereof are provided. The present invention includes a method of employing a plurality of specific primers, and probes to identify the dengue 1 virus, dengue 2 virus, dengue 3 virus, and dengue 4 virus, and a biochip spotted with the specific probes.

Description

200927939 九、發明說明：；【發明所屬之技術領域】 - 本發明是有關於一種鑑定登革病毒的方法，且特別是有關於一種鑑定登革病毒及其血清型的專一性引子組、寡核酸探針、生物晶片及其鑑定方法。【先前技術】200927939 IX. INSTRUCTIONS: [Technical field of invention] - The present invention relates to a method for identifying dengue virus, and in particular to a specific primer set, oligonucleic acid for identifying dengue virus and its serotype Probes, biochips and methods of identification thereof. [Prior Art]

登革熱（dengue fever )與登革出血熱（dengue © hemorrhagical fever)近年的發生有日益嚴重的趨勢，WHO 估計全世界每年約有五千萬人感染登革熱，因此被視為新興傳染病中極重要的「節肢動物攜帶病毒（arbovirus)」傳染病。由於登革熱疫情發生往往會在很短的時間迅速爆發流行，且登革熱患者的病毒血症期（viremia phase )有可能只出現2-3天時間，因此臨床上如何立即、正確診斷為登革病毒感染，成為流行性傳染病防疫工作中非常重要的課題。 ❹ 登革病毒根據抗原性不同，可分為Dengue 1、Dengue 2、Dengue 3、Dengue 4四個血清型。診斷登革熱的方法大致可分病毒分離及血清檢驗二種方法，病毒分離方面，以發病五日内之血清做病毒分離，分離率較高，若超過此 . 期限，由於抗體出現病毒消失，分離不易，此法約需時二週左右；在血清診斷方面，可採病患之急性期及恢復期之血清，藉血球凝集抑制試驗、補體結合試驗及中和試驗測定抗體力價是否有意義上升，然而此種方法不但耗時，更 5 200927939 常因血清採取的時機不適當而影響到結果的判斷。 ' 目前較快速的登革熱診斷方法，已開發出如血清抗體 w 酵素連結免疫分析法（ELIS A )、反轉錄聚合酶連鎖反應 (RT-PCR)分析或即時 RT-PCR (real-time PCR)分析，適用於早期診斷且準確度高，可檢出登革病毒基因，對於登革熱流行區之檢驗助益甚大，但受限於試劑、設備、人員操作技術等因素的影響，不易普及。由於登革熱疫情防治之黃金時期為第一個病例發病〇後一星期内，因此需要發展更精確、快速、方便操作的診斷技術。此外，平時能快速、正確的檢測田間病媒蚊攜帶病毒率，亦可以作為提供疫情預警制度的重要參考。【發明内容】因此本發明就是在提供一種鑑定登革病毒與其血清型之專一性引子（primer)組、募核苷酸探針（probe )及其使用方法，解決傳統登革病毒血清診斷方法耗時且準確度〇不足的的問題。本發明另一方面是在提供一種鑑定登革病毒及其血清型之生物晶片及其使用方法，用以快速鑑定不同血清型之登革病毒。 - 根據本發明，提出一種鑑定登革病毒之專一性引子對，包含 SEQ ID NO :卜 SEQ ID NO : 2、SEQ ID NO : 3、 SEQ ID NO : 4、SEQ ID NO : 5、SEQ ID NO : 6、SEQ ID NO : 7、SEQIDNO ·· 8所示之核苷酸序歹ij、其互補股之核 200927939 苷酸序列、簡併序列及其衍生序列。 ' 根據本發明，提出一種鑑定第一型登革病毒之募核苷 ~ 酸探針及其使用方法，鑑定第一型登革病毒之募核苷酸探針包含 SEQ ID NO ·· 9、SEQ ID NO : 10、SEQ ID NO : 11、 SEQ ID NO : 12、SEQ ID NO : 13、SEQ ID NO : 14、SEQ ID NO : 15、SEQ ID NO : 16所示之核苷酸序列、其互補股之核苷酸序列、簡併序列及其衍生序列。根據本發明，提出一種鑑定第二型登革病毒之寡核苷〇酸探針及其使用方法，鑑定第二型登革病毒之寡核苷酸探針包含 SEQ ID NO : 17、SEQ ID NO : 18、SEQ ID NO : 19、SEQ ID NO : 20、SEQ ID NO : 21、SEQ ID NO : 22、 SEQ ID NO : 23、SEQ ID NO : 24所示之核苷酸序列、其互補股之核苷酸序列、簡併序列及其衍生序列。根據本發明，提出一種鑑定第三型登革病毒之寡核苷酸探針及其使用方法，鑑定第三型登革病毒之寡核苷酸探針包含 SEQ ID NO : 25、SEQ ID NO : 26、SEQ ID NO : 〇 27、SEQ ID NO : 28、SEQ ID NO : 29、SEQ ID NO : 90、 SEQ ID NO : 31、SEQ ID NO : 32所示之核苷酸序列、其互補股之核苷酸序列、簡併序列及其衍生序列。根據本發明，提出一種鑑定第四型登革病毒之募核苷 - 酸探針及其使用方法，鑑定第四型登革病毒之寡核苷酸探，針包含 SEQ ID NO : 33、SEQ ID NO : 34、SEQ ID NO : 35、SEQ ID NO ·· 36、SEQ ID NO : 37、SEQ ID NO : 38、 SEQ ID NO : 39、SEQ ID NO : 40所示之核苷酸序列、其 7 200927939 互補股之核苷酸序列、簡併序列及其衍生序列。 ' 應用上述SEQ ID NO: 9〜40之寡核苷酸探針鑑定登 • 革病毒之方法包含：以登革病毒、登革熱病毒血症患者血液或病媒蚊體萃取之RNA ’進行反轉錄合成cDNA ’再與上述SEQ ID NO ·· 9〜40之募核苷酸探針進行雜合反應，並由雜合反應之結果鑑定登革病毒之及其血清型。根據本發明，提出一種快速鑑定登革熱病毒之生物晶片及其使用方法，鑑定登革病毒之生物晶片包含一基材，〇基材上可固著選自於SEQ ID NO : 9〜40所示之核苷酸序列、其互補股之核苷酸序列、簡併序列、其衍生序列及上述序列之任意組合所組成之族群。應用上述生物晶片鑑定登革病毒之方法包含：以登革病毒、登革熱病毒血症患者血液或感染登革病毒之病媒蚊體萃取之RNA，進行反轉錄合成cDNA，再與上述SEQ ID NO: 9〜40之募核苷酸探針進行雜合反應，並由雜合反應之結果鑑定登革病毒及其血清型。〇本發明之鑑定登革病毒之方法的適用範圍包括登革病毒血清型核酸鑑別、病毒血症患者血液中登革病毒及其血清型檢驗、病媒蚊體登革病毒及其血清型檢驗、細胞培養登革病毒及其血清型檢驗、流行病學調查的快速檢測， - 並適用於各種學術研究單位、疾病管制單位、各型醫院及 ^ 檢驗中心等。根據上述，可知本發明之鑑定登革病毒之方法可達到下列優點： 8 200927939 1.精確檢測：可以精確檢驗登革病毒，同時區分出不 ' 同血清型。 • 2.靈敏檢測：微量病毒即可檢測，靈敏度與反轉錄聚合酶連鎖反應完全一致。 3. 快速檢測：自抽取病毒RNA起，僅需三個半小時，即可獲得具體結果。 4. 操作方便：抽取檢體病毒RNA、反轉錄聚合酶連鎖反應等核酸操作與放大作用均在同一個試管中完成。 © 5.放大的核酸片段只要於晶片上進行雜合作用，即可目視獲知結果，節省進行膠體電泳分析與染色的的麻煩。 6.經濟：僅需要一台聚合酶連鎖反應儀、及一台雜交烘箱設備即可完成操作，設備成本低廉。【實施方式】登革病毒屬於單股正股RNA病毒，核酸長度約11 kb 左右，不同血清型間其序列某些片段變異度頗大，欲選取〇作為鑑定片段極需考量其演化變異度。因此，利用美國國立生物技術信息中心（National Center for Biotechnology Information, NCBI )之基因資料庫，並以台灣周邊國家疫區為基礎，收集四種血清型登革病毒序列，再利用軟體 - Vector NTI® 7.0 ( InforMax Inc·，USA)比對類源關係後，選取共同保守區較高的區域，包括外殼蛋白（Capsid)基因片段、外套膜蛋白（Envelope)基因片段、非結構蛋白基因片段 1 ( Non-structure protein fragment 1，NS1 )和非 9 200927939 結構蛋白基因片段 3 ( Non-structure protein fragment 3， NS3)等序列設計登革病毒專一性引子組，並選取四型病毒之間變異較明顯的區域做為設計募核苷酸探針之對象。再利用原型（prototype )的第一型登革病毒（Dengue 1 virus )、第一型登革病毒（DengUe 2 virus )、第三型登革病毒（Dengue 3 virus )及第四型登革病毒（Dengue 4 vims ) 等不同血清型的登革病毒進行增幅驗證。Dengue fever and dengue hemorrhagical fever have become increasingly serious in recent years. WHO estimates that about 50 million people worldwide are infected with dengue every year, so it is considered to be extremely important in emerging infectious diseases. "Arbovirus" infectious disease. Since the outbreak of dengue fever often occurs rapidly in a short period of time, and the viremia phase of dengue patients may only occur for 2-3 days, how to diagnose dengue virus infection immediately and correctly It has become a very important topic in the epidemic prevention work of epidemic diseases.登 Dengue virus can be divided into four serotypes: Dengue 1, Dengue 2, Dengue 3, and Dengue 4 according to their antigenicity. The method for diagnosing dengue fever can be roughly divided into two methods: virus isolation and serum test. In terms of virus isolation, the virus is separated by serum within five days of onset, and the isolation rate is high. If it exceeds this period, the separation is not easy due to the disappearance of antibodies. This method takes about two weeks; in the serodiagnosis, the serum of the acute and recovery period of the patient can be measured by blood cell agglutination inhibition test, complement fixation test and neutralization test to determine whether the antibody price is meaningfully increased, however, The method is not only time-consuming, but also 5 200927939 often due to the inappropriate timing of the serum to affect the judgment of the results. 'The current rapid dengue diagnostic method has been developed such as serum antibody w enzyme-linked immunoassay (ELIS A), reverse transcriptase polymerase chain reaction (RT-PCR) analysis or real-time PCR analysis. It is suitable for early diagnosis and has high accuracy. The dengue virus gene can be detected. It is very helpful for the detection of dengue endemic areas, but it is not easy to popularize due to factors such as reagents, equipment and personnel operation techniques. Since the golden age of dengue fever prevention and treatment is within one week of the first case, it is necessary to develop diagnostic techniques that are more precise, faster and easier to operate. In addition, the rapid and correct detection of the rate of virus carrying mosquitoes in the field can also be used as an important reference for providing an early warning system for the epidemic. SUMMARY OF THE INVENTION Accordingly, the present invention is to provide a primer set for identifying a dengue virus and a serotype thereof, a probe for raising a nucleotide, and a method for using the same, and solving the problem of the traditional method for diagnosing dengue virus sera. The problem of insufficient accuracy and accuracy. Another aspect of the present invention is to provide a biochip for identifying dengue virus and its serum type and a method of using the same for rapidly identifying dengue viruses of different serotypes. - according to the present invention, a specific primer pair for identifying dengue virus is provided, comprising SEQ ID NO: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO 6. SEQ ID NO: 7, nucleotide sequence 歹 ij represented by SEQ ID NO · 8, nucleus 200927939 nucleotide sequence of its complementary strand, degenerate sequence and its derived sequence. According to the present invention, a nucleoside-to-acid probe for identifying a first type of dengue virus and a method for using the same are provided, and a nucleotide probe for identifying a first type of dengue virus comprises SEQ ID NO. ID NO : 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 nucleotide sequence, complementary thereto Nucleotide sequence, degenerate sequence and derivative sequence thereof. According to the present invention, an oligonucleotide nucleoside probe for identifying a second type of dengue virus and a method for using the same are provided, and the oligonucleotide probe for identifying a second type of dengue virus comprises SEQ ID NO: 17, SEQ ID NO The nucleotide sequence of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, and its complementary strand Nucleotide sequences, degenerate sequences, and derived sequences thereof. According to the present invention, an oligonucleotide probe for identifying a third type of dengue virus and a method for using the same are provided, and the oligonucleotide probe for identifying a third type of dengue virus comprises SEQ ID NO: 25, SEQ ID NO: SEQ ID NO: 〇27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 90, SEQ ID NO: 31, SEQ ID NO: 32, the nucleotide sequence thereof, and its complementary strand Nucleotide sequences, degenerate sequences, and derived sequences thereof. According to the present invention, a nucleoside-acid probe for identifying a fourth type of dengue virus and a method for using the same are provided, and an oligonucleotide probe of a fourth type of dengue virus is identified, the needle comprising SEQ ID NO: 33, SEQ ID Nucleotide sequence of NO: 34, SEQ ID NO: 35, SEQ ID NO. 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, 7 200927939 Nucleotide sequence, degenerate sequence and its derived sequence of the complementary strand. 'Methods for identifying Dengue virus using the oligonucleotide probes of SEQ ID NOs: 9 to 40 above include: reverse transcription synthesis of cDNA by RNA extracted from dengue virus, dengue viremia blood or vector mosquitoes' Further, a heterozygous reaction with the nucleotide probe of the above SEQ ID NO. 9 to 40 was carried out, and the dengue virus and its serotype were identified from the result of the hybrid reaction. According to the present invention, a biochip for rapidly identifying a dengue virus and a method for using the same are provided. The biochip for identifying a dengue virus comprises a substrate, and the substrate can be fixed to be selected from the group consisting of SEQ ID NOS: 9 to 40. A population consisting of a nucleotide sequence, a nucleotide sequence of its complementary strand, a degenerate sequence, a derivative thereof, and any combination of the above sequences. The method for identifying dengue virus by using the above biochip comprises: performing reverse transcription synthesis of cDNA by RNA extracted from dengue virus, dengue viremia patient or pathogen mosquito infected with dengue virus, and then SEQ ID NO: 9~ The nucleotide probe of 40 was subjected to a heterozygous reaction, and the dengue virus and its serotype were identified by the result of the hybrid reaction. The scope of application of the method for identifying dengue virus of the present invention includes dengue virus serotype nucleic acid identification, dengue virus and serotype test in blood of viremia patients, diseased mosquito dengue virus and serotype test thereof, cell culture The rapid detection of dengue virus and its serotype test and epidemiological investigation, and is applicable to various academic research units, disease control units, various hospitals and ^ test centers. According to the above, it can be seen that the method for identifying dengue virus of the present invention can achieve the following advantages: 8 200927939 1. Accurate detection: The dengue virus can be accurately examined, and the same serotype can be distinguished. • 2. Sensitive detection: A small amount of virus can be detected, and the sensitivity is completely consistent with the reverse transcription polymerase chain reaction. 3. Rapid detection: It takes only three and a half hours from the extraction of viral RNA to obtain specific results. 4. Convenient operation: Nucleic acid operation and amplification of sample virus RNA and reverse transcription polymerase chain reaction are performed in the same tube. © 5. Amplified nucleic acid fragments can be visually observed as long as they are hybridized on the wafer, saving the trouble of colloidal electrophoresis analysis and staining. 6. Economy: Only one polymerase chain reaction instrument and one hybrid oven equipment are needed to complete the operation, and the equipment cost is low. [Embodiment] The dengue virus belongs to a single-stranded positive-strand RNA virus. The length of the nucleic acid is about 11 kb. The variability of some fragments of different sequences between different serotypes is quite large. It is necessary to consider the evolution variability of 〇 as the identification fragment. Therefore, using the National Center for Biotechnology Information (NCBI) genetic database and collecting four serotypes of dengue virus sequences based on the epidemic areas around Taiwan, and then using the software - Vector NTI® 7.0 (InforMax Inc., USA) compares the source-like relationship and selects regions with higher conserved regions, including the capsid gene fragment, the envelope protein (Envelope) gene fragment, and the non-structural protein gene fragment 1 (Non -structure protein fragment 1, NS1) and non-9 200927939 Non-structure protein fragment 3 (NS3) sequences were designed to design dengue virus-specific primers, and regions with obvious variation among the four viruses were selected. As a target for the design of nucleotide probes. Reuse the prototype of the first type of Dengue 1 virus, the first type of dengue virus (DengUe 2 virus), the third type of dengue virus (Dengue 3 virus) and the fourth type of dengue virus ( Dengue 4 vims ) The dengue virus of different serotypes was tested for amplification.

依照本發明之實施例，首先利用上述四種血清型登革病毒之外殼蛋白基因、外套膜蛋白基因、非結構蛋白基因片段1 (NS1)和非結構蛋白基因片段3 (NS3)等四個片段設計專一性引子組，包含第一引子組（SEQ ID NO : 1 2)、第二引子組（SEQIDN〇: 3、4)、第三引子組（seq ID NO : 5、6 )及第四引子組（SEQ m N〇 : 7、8 )。請參照第1圖，為本發明之專一性引子組分別應用於四種血清型登革病毒進行增幅之電泳圖。第1圖之（A)為第一引子組與四種血清型登革病毒進仃增幅之電泳圖。第一引子組（SEQ ID N〇 : !、2)為利用四種it清型登革病毒外殼蛋白基因的共同保守區所設計出的專—性引子組，可分別在四種血清型登革病毒樣本中增幅出652驗基對(bP)大小的片段。第1圖（A)中，” M ” 為分子量標記，樣本卜2、3、4分別為利用第一引子組增幅第―、二、三、四型登革病毒外殼蛋白基目之結果，樣本5為空白對照組，用以確^ pcR反應無誤。由第1圖之(A)可看到第一引子組（SEQ出小幻 200927939 可分別在第一、二、 kb大小的片段。第1圖之⑻為第二引子組與四種血清型登革病毒進行增幅之電泳圖。第二引子組（SEQIDNO:3、4)為利用四種血清型登革病毒外套膜蛋白基因的共同保守區所設计出的專-性引子組，可分別在四種血清型登革病毒樣本中增幅出777驗基對⑽大小的片段。第1(B)中，” M” 標記，樣本H、3、4分別為利用第二引子組曰田、—、二、四型登革病毒外套膜蛋白基因之結果，樣本5為空白對照組’用以確定取反應無誤。。八由第1圖之⑻可看到第二引子組(seqidn〇:3、4) 可分別在第二、三、四型登革病#樣本中增幅出約 kb大小的片段。 ❹ 、四型登革病毒樣本中增幅出約〇 65 第1圖之（C)為第三引子組與四種血清型登革病毒進行增幅之電㈣。第HMSEQmN〇:5、6)為利用四種血清型登革病毒非結構蛋白基因片段i (N⑴的共同保守區所設計出的專一性引子組，可分別在四種血清型登革病毒樣本中增幅出419驗基對⑽大小的片段。第 !圖（〇中，” M”為分子量標記，樣本卜2、3、4分別為利用第-引子組增幅第-、二、三、四型登革病毒職基因之-果，樣本5為空白對照組，用以確定pCR反應無誤。。、由第1圖之（C)可看到第三引子組（SEQ ID N〇 : 5、6) 〇另】在第一—、四型登革病毒樣本中增幅出約0.42 kb大小的片段。 11 200927939 第1圖之（D)為第四引子細&π〇搞丄± 行增幅之電泳圖。m 血清型登革病毒進电冰圖。第四引子組（seqidn〇· 7 用四種血清型登革毒 ·、共同保守^蛋白基因片段3(NS3)的呵认彳出的專一性引子組，型登革病純本巾增_ 637 m 财種血/月 + T 637驗基對（bp)大小的片段。八別W圖（D)中，”M”為分子量標記，樣本1、2、3、4 刀别為利用第三引子組增幅第—、二、 NS3基因之紝盅,,,,^ •半两每According to an embodiment of the present invention, four fragments of the above four serotypes of dengue virus coat protein gene, mantle protein gene, non-structural protein gene fragment 1 (NS1) and non-structural protein gene fragment 3 (NS3) are first utilized. Designing a specific primer set comprising a first primer set (SEQ ID NO: 12), a second primer set (SEQIDN〇: 3, 4), a third primer set (seq ID NO: 5, 6), and a fourth primer Group (SEQ m N〇: 7, 8). Referring to Figure 1, the specific primer set of the present invention is applied to the electrophoresis pattern of the amplification of four serotypes of dengue virus. Figure 1 (A) is an electropherogram showing the increase of the first primer set and the four serotypes of dengue virus. The first primer set (SEQ ID N〇: !, 2) is a specialized primer set designed by using the common conserved regions of the four itinergic dengue virus coat protein genes, which can be denatured in four serotypes. A fragment of 652 base pair (bP) size was added to the virus sample. In Fig. 1(A), "M" is the molecular weight marker, and samples 2, 3, and 4 are the results of the amplification of the first, second, third, and fourth types of dengue virus coat protein using the first primer group, respectively. 5 is a blank control group to confirm that the pcR reaction is correct. The first primer set can be seen from (A) of Fig. 1 (SEQ ID NO: 200927939 can be fragmented in the first, second, and kb sizes, respectively. Figure 1 (8) shows the second primer group and the four serotypes. An electrophoretic map of the increase in the virus. The second primer set (SEQ ID NO: 3, 4) is a specialized primer set designed using the common conserved regions of the four serotypes of dengue virus coat protein genes. In the four serotypes of dengue virus samples, the 777 test pair (10) size fragment was increased. In the first (B), the "M" mark, the samples H, 3, and 4 were the second introduction group, the field, -, The results of the second and fourth types of dengue virus mantle membrane protein gene, sample 5 is a blank control group 'to determine the response is correct. Eight by the first figure (8) can see the second introduction group (seqidn〇: 3, 4 Approximately kb-sized fragments can be added to the second, third, and fourth types of dengue disease samples. ❹ and four types of dengue virus samples have an increase of about 65. Figure 1 (C) is the third primer. The group and four serotypes of dengue virus were increased in electricity (IV). HMSEQmN〇: 5, 6) for the use of four serotypes of dengue virus A specific primer set designed by the co-conserved region of the conformon gene fragment i (N(1), which can increase the fragment of 419 test pairs (10) in the four serotypes of dengue virus samples respectively. "M" is the molecular weight marker, and samples 2, 3, and 4 are the results of the first, second, third, and fourth types of dengue virus genes using the first-introduction group, and the sample 5 is a blank control group. The pCR reaction was correct. The third primer set (SEQ ID N〇: 5, 6) was observed from Fig. 1 (C). The increase was about 0.42 in the first- and fourth-type dengue virus samples. Fragment of kb size 11 200927939 (D) is the electrophoresis map of the fourth primer & π 〇丄行行行行行行行。。。。。。。。。 m m m m m m m m m m m m m m 第四第四第四第四第四第四第四第四第四第四第四· 7 The specific priming group of four types of serotypes, dengue, and conserved protein gene fragment 3 (NS3), the type of dengue disease is increased by _ 637 m. T 637 examines the fragment of the (bp) size. In the eight-part W (D), the "M" is the molecular weight marker, and the samples 1, 2, 3, and 4 are used. The three primers increase the number of the first, second, and NS3 genes, and,

因之、-果，樣本5為空白對照組，用以確定pCR反應無誤。。八由第1圖之(D)可看到第三W子組（SEQIDNO:7、8) 可刀別在第一、一、二、四型登革病毒樣本中增幅出約OH kb大小的片段。接著可利用專一性引子組所增幅出之外殼蛋白基因、外套臈蛋白基因、非結構蛋白基因片段i (Nsi)和非結構蛋白基因片段3 (NS3)片段，在四種血清型病毒之間變異較明顯的區域設計專一性寡核苷酸探針，並進行探針之專一性測试之後，得到具有血清型專一性鐘別度之寡核苷酸探針，其序列分別編號為SEQ ID N〇 · 9〜4()。其中編號SEQ ID NO: 9〜10之寡核苷酸探針係利用第一型登革病毒之外殼蛋白基因片段序列設計，可用以鐘定第一型登革病毒。第一型登革病毒之外殼蛋白基因片段序列參見序列識別號碼SEQ ID NO: 41。編號SEQ ID N〇: u 〜12之寡核苷酸探針係利用第一型登革病毒之外套膜蛋白基因片段序列設計，可用以鑑定第一型登革病毒。第一 12 200927939 . 型登革病毒之外套膜蛋白基因片段序列參見序列識別號 W 碼SEQ ID NO: 42。編號SEQ ID NO: 13〜14之募核苷酸探針係利用第一型登革病毒之非結構蛋白基因片段1 (NS 1 )序列設計’可用以鑑定第一型登革病毒。第一型登革病毒之NS1基因片段序列參見序列識別號碼SEQ m NO: 43。編號SEQ ID NO: 15〜16之募核苷酸探針係利用第一型登革病毒之非結構蛋白基因片段3(NS3)序列設 Q 計，可用以鑑定第一型登革病毒。第一型登革病毒之NS3 基因片段序列參見序列識別號碼SEQ ID NO: 44。應說明的是，分別利用SEQ ID NO: 41〜44之全部序列、部分序列或衍生序列所製成之引子或寡核苷酸探針均可用以鑑定第一型登革病毒。其中衍生序列所製成之引子或募核苷酸探針係指一引子或寡核苷酸探針包含一段預定序列’此預定序列包含全部或部分SEQ ID NO : 41〜44 序列的寡核苷酸片段，預定序列含有至少16鹼基（mer)的 ◎ 序列與SEQ ID NO : 41〜44的序列相同》編號SEQ ID NO:17〜18之募核苷酸探針係利用第二型登革病毒之外殼蛋白基因片段序列設計，可用以鑑定第一型登革病毒。第二型登革病毒之外殼蛋白基因片段序列參見序列識別號碼SEQ ID NO: 45 »編號SEQ ID NO: 19 〜20之募核苷酸探針係利用第二型登革病毒之外套膜蛋白基因片段序列設計，可用以鑑定第二型登革病毒。第二型登革病毒之外套膜蛋白1因片&序列I見序列識別號碼SEQ ID NO: 46。編號SEQ ID N〇: 21〜22之募核苷酸 13 200927939 • 探針係利用第二型登革病毒之非結構蛋白基因片段^ w (NS1)序列設計，可用以鑑定第二型登革病毒。第二型登革病毒之NS 1基因片段序列參見序列識別號碼SEq ID NO: 47。編號SEQ ID NO: 23〜24之寡核苷酸探針係利用第二型登革病毒之非結構蛋白基因片段3 ( NS3 )序列設計’可用以鑑定第二型登革病毒。第二型登革病毒之NS3 基因片段序列參見序列識別號碼SEQ ID NO: 48。 Q 分別利用SEQ ID NO : 45〜48之全部序列、部分序列或衍生序列所製成之引子或募核苷酸探針均可用以鑑定第二型登革病毒。其中衍生序列所製成之引子或募核苷酸探針係指一引子或寡核苷酸探針包含一段預定序列，此預定序列包含全部或部分SEQ ID NO: 45〜48序列的寡核苷酸片段’預定序列含有至少16驗基（mer)的序列與SEq ID NO: 45〜48的序列相同。編號SEQ ID NO: 25〜26之寡核苷酸探針係利用第三 ◎ 型登革病毒之外殼蛋白基因片段序列設計，可用以鑑定第二型登革病毒。第三型登革病毒之外殼蛋白基因片段序列參見序列識別號碼SEQ ID NO: 49。編號SEQ ID NO: 27 〜28之募核苷酸探針係利用第三型登革病毒之外套膜蛋白基因片段序列設計，可用以鑑定第三型登革病毒。第三型登革病毒之外套膜蛋白基因片段序列參見序列識別號碼SEQ ID NO: 50。編號SEQ ID NO: 29〜30之募核苷酸探針係利用第三型登革病毒之非結構蛋白基因片段i (NS1 )序列設計’可用以鑑定第三型登革病毒。第三型 200927939 登革病毒之NS1基因片段序列參見序列識別號碼SEq ID NO: 5卜編號SEQ ID NO: 31〜32之寡核苷酸探針係利用第三型登革病毒之非結構蛋白基因片段3 (NS3)序列設计，可用以鑑定第三型登革病毒。第三型登革病毒之NS3 基因片段序列參見序列識別號碼SEQ ID NO: 52。分別利用SEQ ID NO : 49〜52之全部序列、部分序列或衍生序列所製成之引子或寡核苷酸探針均可用以鑑定第三型登革病毒。其中衍生序列所製成之引子或募核苷酸探針係指一引子或募核苷酸探針包含一段預定序列，此預定序列包含全部或部分SEQ ID NO : 49〜52序列的寡核苦酸片段，預疋序列含有至少16驗基（mer)的序列與SEQ ID NO : 49〜52的序列相同。編號SEQ ID NO: 33〜34之募核苷酸探針係利用第四型登革病毒之外殼蛋白基因片段序列設計，可用以鑑定第四型登革病毒。第四型登革病毒之外殼蛋白基因片段序列參見序列識別號碼SEQ ID NO·· 53。編號SEQ ID NO: 35 〜36之寡核苷酸探針係利用第四型登革病毒之外套膜蛋白基因片段序列設計，可用以鑑定第四型登革病毒。第四型登革病毒之外套膜蛋白基因片段序列參見序列識別號碼SEQ ID NO: 54。編號SEQ ID N〇: 37〜38之寡核苷酸探針係利用第四型登革病毒之非結構蛋白基因片段1 (NS1)序列設計，可用以鑑定第四型登革病毒。第四型登革病毒之NS 1基因片段序列參見序列識別號碼SEq NO. 55。編號SEQ ID NO: 39〜40之寡核苷酸探針係利用 15 200927939 . 第四^且革病毒之非結構蛋白基因片段3( NS3 )序列設汁，可用以鑑定第四型登革病毒。第四型登革病毒之NS3 基因片奴序列參見序列識別號碼SEq ID N〇: %。刀別利用SEQ ID NO : 53〜56之全部序列、部分序列或衍生序列所製成之引子或募核苦酸探針均可用以鑑定第四型登革病毒。其中衍生序列所製成之引子或寡核普酸 2針係指—引子或寡㈣酸探針包含-段狀序列，此預冑序列包含全部或部/分SEQ ID NO : 53〜56序列的寡核苷酸片段預疋序列含有至少' i 6驗基（mer)的序列與seq⑴ NO : 53〜56的序列相同。將上述之具有血清型專一性鑑別度之募核苷酸探針 (SEQ ID NO: 9〜40)或上述之預定序列製成生物晶片，可决速鑑疋不同血清型的登革病毒。用以鑑定登革熱病毒之生物晶片包含基材及固著於基材上之寡核苦酸探針，其中土材之材質可包含尼龍膜、高分子材料、石夕片或玻璃。纟物日日片之製作方法包含分別將職莫耳漠度（幽） t合成專一&募核普酸探針溶液分別加入孔盤中，再用點片機點在設定的位置，置於45t烘烤1-2分鐘，最後再以紫外線交聯器（uv_slinker)於08焦爾，6分鐘的條件下將券核苷酸探針固定在晶片上。請參照第2圖，為本發明實施例之生物晶片的寡核苦酸探針施佈示意圖。其中，每一點上所標示之數字代表序列識別編號之數字，數字1〜8代表序列為SEQ ID n〇:9 〜16之寡核苦酸探針，可與含有第—型登革病毒的去氧核 16 200927939 醣核酸樣品產生專一性雜合，而在晶片相對應之位置呈 • 色。標示數字9〜16之點代表序列為SEQ ID NO : 17〜24 ' 之寡核苷酸探針，可與含有第二型登革病毒的去氧核醣核酸樣品產生專一性雜合。標示數字為17〜24之點代表序列分別為SEQ ID NO : 25〜32之寡核苷酸探針，可與含有第三型登革病毒的去氧核醣核酸樣品產生專一性雜合。標示數字為25〜32之點代表序列分別為SEQ ID NO : 33〜40之募核苷酸探針，可與含有第四型登革病毒序列的去氧核醣 © 核酸樣品產生專一性雜合。英文字母，’PC”代表陽性控制組（positive control)，為不同血清型的登革病毒之高度保留區的 DNA 片段 (5，-ATGGGWGARGCAGCHGSDATYTTCATGAC-3，），用以確認樣本萃取及反轉錄聚合酶連鎖反應無誤，字母’’C”則代表控制組，用以確認雜合反應無誤。請參照第3圖，為利用本發明之生物晶片鑑定登革病毒的方法流程圖，包含：〇 ( a )萃取待測樣本之總核醣核酸（total RNA )，其中待測樣本total RNA之來源可為登革病毒、登革病毒病毒血症患者血液或病媒蚊體； (b)進行反轉錄聚合酶連鎖反應（Reverse • transcription-polymerase chain reaction, RT-PCR) > 反轉錄Because of this, the sample 5 was a blank control group to determine that the pCR response was correct. . Eight (D) of Figure 1 shows that the third W subgroup (SEQ ID NO: 7, 8) can increase the fragment size of about OH kb in the first, second, and fourth types of dengue virus samples. . The coat protein gene, the coat prion gene, the non-structural protein gene fragment i (Nsi) and the non-structural protein gene fragment 3 (NS3) fragment, which are amplified by the specific primer set, can then be used to mutate between the four serotype viruses. A more specific region is designed with a specific oligonucleotide probe, and after the specificity test of the probe, an oligonucleotide probe having a serotype specificity is obtained, and the sequences are respectively numbered as SEQ ID N 〇·9~4(). The oligonucleotide probes numbered SEQ ID NOs: 9 to 10 are designed using the sequence of the coat protein gene fragment of the first type of dengue virus, and can be used to determine the first type of dengue virus. Sequence of coat protein gene fragment of type 1 dengue virus See sequence identification number SEQ ID NO: 41. The oligonucleotide probe numbered SEQ ID N: u ~ 12 was designed using the sequence of the outer envelope protein gene fragment of the first type of dengue virus, and can be used to identify the first type of dengue virus. First 12 200927939 . For the dengue virus outer envelope protein gene fragment sequence, see sequence identification number W code SEQ ID NO: 42. The nucleotides of the SEQ ID NO: 13 to 14 probes are designed to identify the first type of dengue virus using the non-structural protein gene fragment 1 (NS 1 ) sequence design of the first type of dengue virus. The sequence of the NS1 gene fragment of the first type of dengue virus is shown in the sequence identification number SEQ m NO: 43. The nucleotide probes of SEQ ID NOs: 15 to 16 are identified by using the non-structural protein gene fragment 3 (NS3) sequence of the first type of dengue virus to identify the first type of dengue virus. The sequence of the NS3 gene fragment of the first type of dengue virus is shown in SEQ ID NO: 44. It should be noted that primers or oligonucleotide probes prepared using all of the sequences, partial sequences or derived sequences of SEQ ID NOS: 41 to 44, respectively, can be used to identify the first type of dengue virus. A primer or a nucleotide probe prepared by the derived sequence means that a primer or an oligonucleotide probe comprises a predetermined sequence of oligonucleosides comprising a sequence of all or part of SEQ ID NO: 41 to 44. The acid fragment, the predetermined sequence contains at least 16 bases (mer), the sequence is identical to the sequence of SEQ ID NOs: 41 to 44. The nucleotide probes of the numbered SEQ ID NOs: 17 to 18 utilize the second type of dengue. The viral coat protein gene fragment sequence is designed to identify the first type of dengue virus. Sequence of the coat protein gene fragment of the second type of dengue virus, see sequence identification number SEQ ID NO: 45 » number SEQ ID NO: 19 to 20 of the nucleotide probes using the second type of dengue virus The fragment sequence is designed to identify a second type of dengue virus. The second type of dengue virus is a membrane protein 1 due to the fragment & sequence I see sequence identification number SEQ ID NO: 46. No. SEQ ID N〇: 21~22 nucleotides 13 200927939 • The probe system is designed using the second type of dengue virus non-structural protein gene fragment w (NS1) sequence, which can be used to identify the second type of dengue virus . The sequence of the NS 1 gene fragment of the second type of dengue virus is shown in the sequence identification number SEq ID NO: 47. The oligonucleotide probes numbered SEQ ID NOs: 23 to 24 can be used to identify the second type of dengue virus using the non-structural protein gene fragment 3 (NS3) sequence design of the second type of dengue virus. The sequence of the NS3 gene fragment of the second type of dengue virus is shown in SEQ ID NO: 48. A primer or a nucleotide probe prepared by using all of the sequences, partial sequences or derived sequences of SEQ ID NOs: 45 to 48, respectively, can be used to identify the second type of dengue virus. A primer or a nucleotide probe prepared by the derived sequence means that a primer or an oligonucleotide probe comprises a predetermined sequence comprising all or part of the oligonucleosides of SEQ ID NO: 45 to 48. The sequence of the acid fragment 'predetermined sequence containing at least 16 mers (mer) is identical to the sequence of SEq ID NO: 45 to 48. The oligonucleotide probes of the SEQ ID NOs: 25 to 26 are designed using the coat protein gene fragment sequence of the third type of dengue virus, and can be used to identify the second type of dengue virus. Sequence of the coat protein gene fragment of the third type of dengue virus See sequence identification number SEQ ID NO: 49. The nucleotide probes of SEQ ID NO: 27 to 28 were designed using the sequence of the outer envelope protein gene of the third type of dengue virus and can be used to identify the third type of dengue virus. The sequence of the envelope protein gene fragment of the third type of dengue virus is shown in the sequence identification number SEQ ID NO: 50. Nucleotide Nos. SEQ ID NO: 29 to 30 The probe is designed to recognize the third type of dengue virus using the non-structural protein gene fragment i (NS1) sequence design of the third type of dengue virus. Type III 200927939 NS1 gene fragment sequence of dengue virus See sequence identification number SEq ID NO: 5 SEQ ID NO: 31 to 32 oligonucleotide probes using a non-structural protein gene of the third type of dengue virus Fragment 3 (NS3) sequence design can be used to identify type III dengue virus. The sequence of the NS3 gene fragment of the third type of dengue virus is shown in the sequence identification number SEQ ID NO: 52. Primers or oligonucleotide probes made using the entire sequence, partial sequence or derived sequence of SEQ ID NOS: 49-52, respectively, can be used to identify the third type of dengue virus. A primer or a nucleotide probe prepared by the derivative sequence means that a primer or a nucleotide probe comprises a predetermined sequence comprising all or part of the oligonucleotide of SEQ ID NO: 49 to 52. The acid fragment, the sequence of the pre-sequence containing at least 16 mer (mer) is identical to the sequence of SEQ ID NO: 49-52. The nucleotide probes of SEQ ID NOs: 33 to 34 are designed using the coat protein gene fragment sequence of the fourth type of dengue virus and can be used to identify the fourth type of dengue virus. Sequence of coat protein gene fragment of type 4 dengue virus See sequence identification number SEQ ID NO..53. The oligonucleotide probes numbered SEQ ID NO: 35 to 36 were designed using the sequence of the outer envelope protein gene of the fourth type of dengue virus and can be used to identify the fourth type of dengue virus. The sequence of the envelope protein gene fragment of the fourth type of dengue virus is shown in the sequence identification number SEQ ID NO: 54. Nucleotide No. SEQ ID N: 37~38 The probe was designed using the non-structural protein gene fragment 1 (NS1) sequence of the fourth type of dengue virus and can be used to identify type 4 dengue virus. The sequence of the NS 1 gene fragment of the fourth type of dengue virus is shown in the sequence identification number SEq NO. 55. The oligonucleotide probes of SEQ ID NO: 39 to 40 are used to identify the type 4 dengue virus by using the non-structural protein gene fragment 3 (NS3) sequence of the fourth virus. For the NS3 gene fragment sequence of the fourth type of dengue virus, see the sequence identification number SEq ID N〇: %. A primer or a nucleus acid probe prepared by using all sequences, partial sequences or derived sequences of SEQ ID NOS: 53 to 56 can be used to identify type 4 dengue virus. Wherein the primer or the oligonucleotide 1 needle referred to in the derived sequence comprises a primer or an oligo (tetra) acid probe comprising a segment sequence comprising all or part of the sequence of SEQ ID NO: 53 to 56 The oligonucleotide fragment pre-sequence contains a sequence of at least 'i 6 mer (mer) which is identical to the sequence of seq(1) NO: 53-56. The above-mentioned nucleotide probes (SEQ ID NOS: 9 to 40) having the serotype specificity discrimination or the predetermined sequence described above can be made into a biochip, and the dengue viruses of different serotypes can be determined quickly. The biochip for identifying dengue virus comprises a substrate and an oligonucleotide probe immobilized on the substrate, wherein the material of the soil material may comprise a nylon membrane, a polymer material, a stone tablet or a glass. The production method of the scorpion day film includes separately adding the Momo Moment (Secret) t synthesis specific & nucleotide probe solution to the orifice plate, and then placing the spot machine at the set position, placing Bake for 1-2 minutes at 45t, and finally fix the coupon nucleotide probe to the wafer with a UV crosslinker (uv_slinker) at 08 Jol for 6 minutes. Referring to Figure 2, there is shown a schematic diagram of the implantation of an oligonucleotide probe of a biochip according to an embodiment of the present invention. Wherein, the number indicated on each point represents the number of the sequence identification number, and the numbers 1 to 8 represent the oligonucleotide probe of the sequence of SEQ ID n〇: 9 to 16, which can be combined with the type-containing dengue virus. Oxygen nucleus 16 200927939 A sample of a sugar nucleic acid produces a specific hybrid, which is colored at the corresponding position of the wafer. The dot numbered 9 to 16 represents an oligonucleotide probe having the sequence of SEQ ID NO: 17 to 24', which can be specifically hybridized with a sample of a deoxyribonucleic acid containing a second type of dengue virus. The dot number indicating 17 to 24 represents an oligonucleotide probe of SEQ ID NO: 25 to 32, respectively, which can be specifically hybridized with a DNA sample containing a third type of dengue virus. The point indicated by the number 25 to 32 represents a nucleotide probe of SEQ ID NO: 33 to 40, respectively, which can be specifically hybridized with a deoxyribose-DNA sample containing a fourth type of dengue virus sequence. The English letter, 'PC' stands for positive control, is a highly retained DNA fragment of dengue virus of different serotypes (5,-ATGGGWGARGCAGCHGSDATYTTCATGAC-3), used to confirm sample extraction and reverse transcriptase polymerase The chain reaction is correct, and the letter ''C' stands for the control group to confirm that the hybrid reaction is correct. Referring to FIG. 3, a flow chart of a method for identifying dengue virus using the biochip of the present invention comprises: 〇(a) extracting total RNA of a sample to be tested, wherein the source of the sample to be tested is available. For dengue virus, dengue virus viremia patients, blood or vector mosquitoes; (b) reverse transcription-polymerase chain reaction (RT-PCR) > reverse transcription

. total RNA並增幅互補去氧核醣核酸（cDNA)。其中，RT-PCR 係採取多目標PCR ( Multiplex-PCR )模式’加入四組PCR 引子對混合在一起使用； 17 200927939 (C)使用本發明之生物晶片與增幅之cDNA片段進行雜合反應，以及 v ( d)鑑定步驟（c)之雜合反應結果，依照色斑之有無鑑定登革病毒之血清型。依照本發明之實施例，萃取待測樣本之total RNA的方法包含利用 QIAamp viral RNA kit (QIAGEN，CA USA)，並根據其說明書進行萃取。將萃取的total RNA反轉錄合成cDNA的方法係利用 ® Superscript one-step RT-PCR kit ( Invitrogen, Carlsbad, CA USA)並依其步驟進行反應。利用多目標PCR模式，將四組專一性引子組，包括第一引子組（SEQ ID NO : 1、2 )、第二引子組（SEQ ID NO: 3、4)、第三引子組（SEQ ID NO : 5、6)及第四引子組（SEQ ID NO : 7、8)混合在一起使用，並分別以不同血清型登革病毒萃取的total RNA來增幅cDNA。聚合酶連鎖反應係以聚合酶連鎖反應儀進行，利用具有生物素（Biotin)標定的引子進行聚合酶連鎖反 Ο 應，可得到具有生物素標定的cDNA片段，即為雜合反應之標的（target)。反應後之產物可進行雜合反應》將生物素標定的target片段與如第2圖所示之本發明的生物晶片上之募核苷酸探針進行雜合。依照本發明之實施 * 例，進行雜合反應之方法包含在晶片每一反應槽先加入 . 200微升（μΐ)雜合反應液’再加入適量經94°C 5分鐘加熱變性之生物素標定的target片段與之混合，置於43°C環境下 1小時，以進行雜合反應。之後以200 μΐ的緩衝清洗液 18 200927939 (washing buffer)分別清洗3次，洗去未雜合的target片段》再加入200私1的阻斷劑（blocking reagent ;含有0.2 jLtl v streptavidin conjugated alkaline phosphatase, Strep-AP )反應30分鐘，再以200 μΐ緩衝清洗液清洗3次，加入200 μΐ 顯色劑（4 μΙΝΒΤ/BCIP與 196 μΐ detection buffer)於晶片反應槽中’置於暗室反應5分鐘，隨即呈色。反應後以清水清洗並烘乾’以出現色斑的有無判定登革病毒的種類。請參照第4圖’為利用第一型登革病毒之cDNA樣本 ® 測試本發明之生物晶片之探針的專一性結果。對照第2圖之募核苷酸探針施佈示意圖，以第一型登革病毒之cDNA 樣本與本發明之生物晶片上之募核苷酸探針雜合的結果顯示’試驗組只有第1〜8號的點有呈色，陽性控制組（PC ) 及控制組（C)亦有呈色，故可確認RNA萃取、RT-PCR 反應及雜合反應均無誤。其中，1〜8點代表序列為SEQ ID NO: 9〜16之寡核苷酸探針，因此SEQ ID NO : 9〜16之寡核苷酸探針可與含有第一型登革病毒之樣品產生專一 © 性雜合，可用以準確鑑定第一型登革病毒。請參照第5圖，為利用第二型登革病毒之cDNA樣本測試本發明之生物晶片之探針的專一性結果。對照第2圖之寡核苷酸探針施佈示意圖，以第二型登革病毒之cDNA ' 樣本與本發明之生物晶片上之募核苷酸探針雜合的結果 • 顯示，試驗組只有第9〜16號的點有呈色，陽性控制組（PC )Total RNA and amplification of complementary deoxyribonucleic acid (cDNA). Wherein, RT-PCR is performed by using a multi-target PCR (multiplex-PCR) mode 'addition of four sets of PCR primer pairs; 17 200927939 (C) using the biochip of the present invention to hybridize with the amplified cDNA fragment, and v (d) Identify the results of the heterozygous reaction of step (c) and identify the serotype of dengue virus according to the presence or absence of stains. According to an embodiment of the present invention, the method of extracting the total RNA of the sample to be tested comprises using QIAamp viral RNA kit (QIAGEN, CA USA) and performing extraction according to the instructions thereof. The method of reverse transcription synthesis of cDNA from extracted total RNA was carried out according to the procedure using a ® Superscript one-step RT-PCR kit (Invitrogen, Carlsbad, CA USA). Using the multi-target PCR model, four sets of specific primers, including the first primer set (SEQ ID NO: 1, 2), the second primer set (SEQ ID NO: 3, 4), and the third primer set (SEQ ID) NO: 5, 6) and the fourth primer set (SEQ ID NO: 7, 8) were used in combination, and the total RNA was amplified by different serotypes of dengue virus-extracted total RNA. The polymerase chain reaction is carried out by a polymerase chain reaction reactor, and a biotin-labeled cDNA fragment is obtained by using a biotin-labeled primer to obtain a biotin-labeled cDNA fragment, which is a target of a hybrid reaction (target). ). The product after the reaction can be subjected to a hybrid reaction. The biotin-labeled target fragment is hybridized with the nucleotide probe on the biochip of the present invention as shown in Fig. 2. According to an embodiment of the present invention, the method for performing the hybrid reaction comprises first adding 200 microliters (μΐ) of the hybrid reaction solution in each reaction tank of the wafer, and then adding an appropriate amount of biotin calibration by heat denaturation at 94 ° C for 5 minutes. The target fragment was mixed with it and placed in an environment of 43 ° C for 1 hour to carry out a heterozygous reaction. Then, wash twice with 200 μΐ buffer cleaning solution 18 200927939 (washing buffer), wash away the unhybrid target fragment, and add 200 blocking reagent (containing 0.2 jLtl v streptavidin conjugated alkaline phosphatase, Strep-AP) was reacted for 30 minutes, then washed 3 times with 200 μM buffer solution, and 200 μΐ of color developer (4 μΙΝΒΤ/BCIP and 196 μΐ detection buffer) was added to the reaction chamber for 5 minutes in the dark reaction chamber. Coloring. After the reaction, it was washed with water and dried to determine the type of dengue virus by the presence or absence of stain. Please refer to Fig. 4' for the specificity results of the probe of the biochip of the present invention using the cDNA sample ® of the first type of dengue virus. According to the schematic diagram of the nucleotide probe of Figure 2, the result of hybridization of the cDNA sample of the first type of dengue virus with the nucleotide probe on the biochip of the present invention shows that the test group has only the first The dots of ~8 have coloration, and the positive control group (PC) and the control group (C) also have coloration, so it can be confirmed that the RNA extraction, RT-PCR reaction and hybrid reaction are correct. Wherein, 1 to 8 points represent oligonucleotide probes of the sequence of SEQ ID NOS: 9 to 16, so that the oligonucleotide probes of SEQ ID NOS: 9 to 16 can be sampled with the first type of dengue virus Produce a specific heterozygosity that can be used to accurately identify the first type of dengue virus. Referring to Figure 5, the specificity results of the probe of the biochip of the present invention were tested using a cDNA sample of the second type of dengue virus. The results of the oligonucleotide probe of Figure 2 are compared with the results of the hybridization of the cDNA sample of the second type of dengue virus with the nucleotide probe on the biochip of the present invention. Points from 9th to 16th have coloration, positive control group (PC)

及控制組（C )亦有呈色，故可確認RNA萃取、RT-PCR 反應及雜合反應均無誤。其中，9〜16點代表序列為SEQ ID 19 200927939 NO : 17〜24之募核苷酸探針，因此SEQ ID NO ·· 17〜24 * 之寡核苷酸探針可與含有第二型登革病毒之樣品產生專 ' 一性雜合，可用以準確鑑定第二型登革病毒。And the control group (C) also showed color, so it was confirmed that the RNA extraction, RT-PCR reaction and hybrid reaction were correct. Wherein, 9 to 16 points represent a nucleotide probe of the sequence of SEQ ID 19 200927939 NO: 17 to 24, and thus the oligonucleotide probe of SEQ ID NO 17 17 can be associated with the second type Samples of the leather virus produce a 'sexual heterozygosity that can be used to accurately identify the second type of dengue virus.

請參照第6圖，為利用第三型登革病毒之cDNA樣本測試本發明之生物晶片之探針的專一性結果。對照第2圖之募核苷酸探針施佈示意圖，以第三型登革病毒之cDNA 樣本與本發明之生物晶片上之募核苷酸探針雜合的結果顯示，試驗組只有第17〜24號的點有呈色，陽性控制組〇 ( PC )及控制組（C )亦有呈色，故可確認RNA萃取、RT-PCR 反應及雜合反應均無誤。其中，17〜24點代表序列為SEQ ID NO : 25〜32之募核苷酸探針，因此SEQ ID NO : 25〜 32之募核苷酸探針可與含有第三型登革病毒之樣品產生專一性雜合，可用以準確鑑定第三型登革病毒。請參照第7圖，為利用第四型登革病毒之cDNA樣本測試本發明之生物晶片之探針的專一性結果。對照第2圖之募核苷酸探針施佈示意圖，以第四型登革病毒之cDNA 〇樣本與本發明之生物晶片上之寡核苷酸探針雜合的結果顯示，試驗組只有第25〜32號的點有呈色，陽性控制組 (PC)及控制組（C)亦有呈色，故可確認RNA萃取、RT-PCR 反應及雜合反應均無誤。其中，25〜32點代表序列為SEQ • ID NO : 33〜40之寡核苷酸探針，因此SEQ ID NO : 33〜 . 40之募核苷酸探針可與含有第四型登革病毒之樣品產生專一性雜合，可用以準確鑑定第四型登革病毒。依照本發明之實施例，本發明之生物晶片可包含上述 20 200927939 SEQ ID NO : 9〜40所示之核苷酸序列、其互補股之核苦酸序列、簡併序列及其衍生序列。此處所指之衍生序列係 ' 於序列SEQ ID NO: 9〜40之3’端或5,端修飾核苦酸序列，使其仍和原序列具有70%以上相似性之募核皆酸序列。雖然本發明已以數實施例揭露如上，然其並非用以限定本發明，任何熟習此技藝者，在不脫離本發明之精神和範圍内’當可作各種之更動與潤飾，因此本發明之保護範圍當視後附之申請專利範圍所界定者為準。〇【圖式簡單說明】為讓本發明之上述和其他目的、特徵、優點與實施例能更明顯易懂，所附圖式之詳細說明如下：第1圖為本發明之專一性引子組分別應用於四種血清蜇登革病毒進行增幅之電泳圖。第2圖為本發明之生物晶片的寡核苷酸探針施佈示意〇圖。第3圖係繪示依照本發明實施例的一種以生物晶片鑑定登革病毒的方法流程圖。第4圖為利用第一型登革病毒之eDNA樣本測試本發 - 明之生物晶片之探針的專一性結果。 • 第5圖為利用第二型登革病毒之cDNA樣本測試本發明之生物晶片之探針的專一性結果。第6圖為利用第三型登革病毒之cDNA樣本測試本發 21 200927939 ❹Referring to Figure 6, the specificity results of the probe of the biochip of the present invention were tested using a cDNA sample of the third type of dengue virus. According to the schematic diagram of the nucleotide probe of Figure 2, the cDNA sample of the third type of dengue virus was hybridized with the nucleotide probe on the biochip of the present invention, and the test group only had the 17th. The dots of ~24 have coloration, and the positive control group (PC) and the control group (C) also have coloration, so it can be confirmed that the RNA extraction, RT-PCR reaction and hybrid reaction are correct. Wherein, 17 to 24 points represent a nucleotide probe of the sequence of SEQ ID NO: 25 to 32, and thus the nucleotide probe of SEQ ID NO: 25 to 32 can be used with a sample containing the third type of dengue virus. Produce specific heterozygous, which can be used to accurately identify the third type of dengue virus. Referring to Figure 7, the specificity results of the probe of the biochip of the present invention were tested using a cDNA sample of the fourth type of dengue virus. According to the schematic diagram of the nucleotide probe of Figure 2, the result of hybridization of the cDNA sample of the fourth type of dengue virus with the oligonucleotide probe of the biochip of the present invention shows that the test group has only the first The dots from 25 to 32 have coloration, and the positive control group (PC) and the control group (C) also have coloration, so it is confirmed that the RNA extraction, the RT-PCR reaction, and the hybrid reaction are correct. Wherein, 25 to 32 points represent an oligonucleotide probe having the sequence of SEQ • ID NO: 33 to 40, and thus the nucleotide probe of SEQ ID NO: 33 to 40 may be associated with the fourth type of dengue virus. The sample produces a specific hybrid that can be used to accurately identify type 4 dengue virus. According to an embodiment of the present invention, the biochip of the present invention may comprise the nucleotide sequence shown in SEQ ID NO: 9 to 40 of the above-mentioned 20 200927939, the nucleotide sequence of the complementary strand, the degenerate sequence, and the derivative sequence thereof. The derivative sequence referred to herein is a nucleotide sequence which is at the 3' end of the sequence of SEQ ID NOs: 9 to 40 or 5, which modifies the nucleotide sequence to have a similarity to the original sequence of 70% or more. The present invention has been disclosed in the above embodiments, and is not intended to limit the invention. It is to be understood that the invention may be modified and modified without departing from the spirit and scope of the invention. The scope of protection is subject to the definition of the scope of the patent application attached. BRIEF DESCRIPTION OF THE DRAWINGS The above and other objects, features, advantages and embodiments of the present invention will become more <RTIgt; It is applied to the electrophoresis pattern of four serum sputum dengue viruses for amplification. Fig. 2 is a schematic view showing the implantation of an oligonucleotide probe of the biochip of the present invention. Figure 3 is a flow chart showing a method for identifying a dengue virus using a biochip according to an embodiment of the present invention. Figure 4 is a graph showing the specificity of the probe of the biochip of the present invention using the eDNA sample of the first type of dengue virus. • Figure 5 is a specific result of testing the probe of the biochip of the present invention using a cDNA sample of the second type of dengue virus. Figure 6 shows the test using the cDNA sample of the third type of dengue virus. 21 200927939 ❹

明之生物晶片之探針的專一性結果。第7圖為利用第四型登革病毒之cDNA樣本測試本發明之生物晶片之探針的專一性結果。【主要元件符號說明】無 22The specificity of the probe of the biochip. Figure 7 is a graph showing the specificity of the probe of the biochip of the present invention using a cDNA sample of the fourth type of dengue virus. [Main component symbol description] None 22

Claims

200927939 X. Patent Application Range: 1. An oligonucleotide probe for identifying dengue virus, which is selected from the nucleotide sequence shown by SEQ ID NO: 9-40, and its complementary strand nucleoside A group consisting of an acid sequence, a degenerate sequence, and a derivative thereof. 2. An oligonucleotide probe for identifying a dengue virus according to the scope of the patent application, wherein the derivative sequence is represented by the sequence SEQidn: ❹

9, 10, U, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 3, 32, 33 '34, 35, 36 37, 38, 39 or 40 The 3' or 5' end of the nucleotide sequence is modified such that it still has 70% or more similarity to the original sequence. 3. A method for identifying a dengue virus comprising: (a) extracting a total ribonucleic acid of a sample to be tested; (b) performing a reverse transcription polymerase chain reaction, a reverse transcription step (& product and increasing complementary deoxygenation) Ribonucleic acid; (1) using a nucleoside acid probe to carry out a hybrid reaction with the product of step (1), wherein the nucleus (four) probe is selected from the nucleotide sequence shown by SEQ m N〇: 9 to 40, a group consisting of a nuclear (four) sequence of complementary strands, a degenerate sequence and a sequence derived therefrom; and U) a heterozygous reaction result of the identification step (1), identifying the serotype of the dengue virus according to the presence or absence of the stain. 4. If the patent application scope is as described in item 3, the method of dengue virus is specified, 23 200927939 = sampler:::: source can be dengue ", ... 5. as claimed in the third paragraph of the patent application scope A method of leather virus, wherein the reverse transcription polymerase chain reaction adopts a multi-target PCR mode.

❹ 6· The method for identifying dengue virus according to claim 5 of the patent scope, wherein the multi-target PCR (4) specific-specific primer group comprises the first group (SEQ ID N〇: 1, 2), the second primer Group (SEQ ID NO. 34), third primer set (SEQ IDN: 5'6) and fourth primer ID NO: 7, 8). ^ 7. The method of claiming dengue virus according to item 3 of the patent application, wherein the derived sequence is referred to in the sequence of SEQ ID NO: 9, 1, u, u, 12 13, 14, 15, 17, 18, 19 , 2〇, 21, 22, 23, 24 25, , : 7, 28” 9, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or. The nucleotide sequence of the 3' terminus or 5' terminus of 40 is such that it still has 70% or more similarity to the original sequence. 8. The method for identifying dengue virus as described in claim 3, further comprising labeling the product of step (b) with a labeling molecule. 9. Method for identifying dengue virus as described in claim 8 Wherein the labeled molecule is labeled Biotin. 24 Ο Ο 200927939 ϊ〇. A biochip for identifying dengue virus, comprising a substrate; and: an oligonucleotide probe, affixed to the substrate In the material, the (tetra) acid probe is selected from the group consisting of the nucleotide sequence shown by SEQ ID NOS: 9 to 40, the nucleus (four) (four), the (four) phase of the complementary strand, the derivative sequence thereof, and any combination of the above sequences. U. The biochip for dengue virus as described in the scope of claim K) wherein the derived sequence is referred to in the sequence of SEQ ID NO: 9, 1, 、, n, 12, 13, 14, 15 , 17, ls, i9, 2〇2i, 2223, 24 \ 25 ' 26 ' 27 ' 28 ' 29, 3G, 31, 32, 33, 34, 35, 36, 39 or 40 3' end or 5, end The nucleotide sequence of the nucleotide sequence is modified so that it has a similarity to the original sequence of 7 % or more. The tablet for identifying a dengue virus, wherein the material of the substrate comprises a nylon film, a polymer material, a batt or a glaze, and the biochip according to claim 10 of the patent application scope is used for dengue The method of virus comprises: () extracting the total ribonucleic acid of the sample to be tested; t (b) performing a reverse transcription polymerase chain reaction, reverse transcription of the product of step (a) and increasing the complementary deoxyribonucleic acid; 25 200927939 and The step-release spot has (7) the hybridization reaction using the product of the biochip (b) described in claim 9 and (4) the step (the result of the heterozygous reaction, the serotype of the dengue virus is not identified according to the color 2. The method for identifying dengue disease by using a biochip as described in claim 13 wherein the source of the total ribozyme nucleic acid of the sample to be tested in step (c) may be a dengue virus or a dengue viremia patient. Gold liquid or vector-derived body. 15. A method for identifying a dengue virus using a biochip as described in claim 13 wherein the reverse transcription polymerase chain reaction of step (c) adopts a multi-target j&gt The CR mode. The method of identifying a dengue virus using a biochip as described in claim 15 wherein the specific primer set used in the multi-target PCR comprises a first primer set (SEQ ID NO: 1, 2) ), a second primer set (SEQ ID NO: 3, 4), a third primer set (seQ ID NO ··5, 6), and a fourth primer set (SEQ ID NO: 7, 8). i7. The method of identifying a dengue virus using a biochip as described in item 13 further comprises labeling the product of step (b) with a labeling molecule. 26 200927939 18. A method for identifying a dengue virus using a biochip as described in claim 17 of the patent scope wherein the labeling molecule is biotin. 19. A dengue virus identification kit comprising at least one specific primer set, wherein the one-specific primer set is selected from the nucleotide sequence of the complementary strand of the nucleotide sequence represented by seq1NO: i8, A group consisting of a sequence, a sequence of any of the sequences, and any combination of the above sequences. 20. A dengue virus identification kit comprising at least one nucleotide probe selected from the group consisting of nucleotide sequences not identified by SEQ ID N: 9-40 A group consisting of a nucleotide sequence of its complementary strand, a degenerate sequence, a derivative thereof, and any combination of the above sequences. An oligonucleotide fragment for identifying a first type of dengue virus, comprising a predetermined sequence, and the entire sequence or portion of the oligonucleotide fragment having a serotype-specific discrimination The sequences are identical, wherein the oligonucleotide fragment having the serotype specificity is selected from the sequence identification numbers SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44. The group of people. 22. A nucleotide fragment for use in identifying a first type of dengue virus as set forth in claim 21, wherein the predetermined sequence comprises at least 16 bases. 23. As identified in claim 21, the first type of dengue disease 27 200927939 toxic nucleotide fragment is used, wherein the predetermined sequence is used as an oligonucleic acid probe. 24. An oligonucleotide fragment for identifying a second type of dengue virus comprising: a predetermined sequence of the predetermined sequence and a sequence or portion of a nucleotide sequence having a serotype specificity The sequences are identical, wherein the oligonucleotide fragment having the serotype specificity is selected from the sequence identification numbers SEQ ID NO: 45 ' SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48 The group of people. 25. A nucleotide fragment for use in the identification of a second type of dengue virus as set forth in claim 24, wherein the predetermined sequence comprises at least 16 bases. 26. A nucleotide fragment for use in identifying a second type of dengue virus as claimed in claim 24 wherein the predetermined sequence is used as a nucleoside probe. 27. An oligonucleotide fragment for identifying a third type of dengue virus, comprising a predetermined sequence and a sequence or portion of an oligonucleotide fragment having a serotype-specificity discrimination The sequence is the same, wherein the nucleotide fragment having the serotype specificity is selected from the sequence identification numbers SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52. The group of people. 28 200927939 28. A nucleotide fragment for the identification of a third type of dengue virus, as set forth in claim 27, wherein the predetermined sequence comprises at least 16 bases. 29. A nucleotide fragment for the identification of a third type of dengue disease, as claimed in claim 27, wherein the predetermined sequence is used as a nucleoside probe. An oligonucleotide fragment for identifying a fourth type of dengue virus, comprising a predetermined sequence and a sequence represented by an oligonucleotide fragment having a serotype-specific discrimination or The partial sequences are identical, wherein the oligonucleotide fragment having the serotype specificity is selected from the sequence identification numbers SEQ ID NO: 53, SEq ID: 54, SEQ m N〇: 55, and SEQ ID NO: 56. The group of people formed. 31. A nucleotide fragment for use in the identification of a fourth type of dengue virus, as set forth in claim 30, wherein the predetermined sequence comprises at least 16 bases. 32. A nucleotide fragment for identifying a type 4 dengue virus as claimed in claim 30, wherein the predetermined sequence is used as a nucleoside 29 200927939 <110> National Chung Hsing University <120> A specific primer set, a nucleotide probe, a biochip, and an identification method thereof for identifying dengue virus <160 >56 <210> SEQ ID NO: 1 <211>21 <212>DNA <213> Artificial sequence < 220 > Primer bind <223> The coat protein gene primer of dengue virus <400>1 aatatgctga aacgcghgag a 21 Q <210> SEQ ID NO: 2 <211>19 <212>DNA <213>Artificialsequence<220> Primer bind <223> The coat protein gene primer of dengue virus <400 >2 argcbccttc hgmbgacat 19 ❹ <210> SEQ ID NO: 3 <211>21 <212>DNA <213>Artificial sequence. <220 > Primer bind <223> Dengue virus envelope protein gene primer <400>3 atcncargar ggrgchatgc a 21 30 200927939 <210> SEQ ID NO: 4 <211><212>DNA<213> artificial sequence < 220 > Primer bind <223> Dengue virus envelope protein gene primer <400>4

Crcctwccac atttbarttc t 21 <210> SEQ ID NO: 5 <211>20 <212>DNA<213>Artificial sequence<220> Primer bind <223> Dengue virus non-structural protein gene 1 (SN1 )Introduction<400>5 ❹ gayatgggbt aytggataga 20 <210> SEQ ID NO: 6 <211>20 <212>DNA <213>Artificial Sequence<220 > Primer bind <223> Structural protein gene 1 (SN1) primer 31 200927939 <400>6 ctratytcca tvccrtacca 20 <210> SEQ ID NO: 7 <211>21 <212>DNA <213>Artificial sequence<220 > Primer bind Q &lt ;223> Non-structural protein gene 3 (SN3) primer of dengue virus <400>7 tvatggatga rgcmcatttc a 21 <210> SEQ ID NO: 8 <211>20 <212>DNA <213 > Artificial sequence < 220 > Primer bind <223 > Non-structural protein gene 3 (SN3) primer of dengue virus <400 >8 agcatyttwg cttctktcca 20 <210> SEQ ID NO: 9 <211>23 <212> DNA 32 200927939 <213>Artificial sequence <220><223> Oligonucleotide probe for type 1 dengue virus <400>9 gaatggagcg atcaaagtgt tac 23

<210> SEQ ID NO: 10 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 >< 223 > Identification of oligonucleotide probe of the first type of dengue virus <400 &gt ; 10 tctgtgacca tgctccttat 20 <210> SEQ ID NO: 11 <211 > 22 Ο <212>DNA <213 > Artificial Sequence <220>< 223 > Identification of the first type of dengue virus Glycoside probe <400>11 gagaaaggag taacccagaa tg 22 <210> SEQ ID NO: 12 <211>22 33 200927939 <212>DNA <213 > artificial sequence <220><223> Oligonucleotide probe for dengue virus <400 >12 attaaactca aggagcacgt cc 22

<210> SEQ ID NO: 13 < 211 > 23 < 212 > DNA < 213 > Artificial Sequence < 220 >< 223 > Identification of the first type of dengue virus nucleotide probe <400 &gt ; 13 tatatggagg accaatatct cag 23 <210>SEQ ID NO: 14 <211>22 ❹ <212>DNA <213 > Artificial sequence <220><223> Identification of the first type of dengue virus Nucleotide probe <400 >14 agtcacagga aagataatcc at 22 <210> SEQ ID NO: 15 34 200927939 <211>18 <212>DNA <213 > artificial sequence <220><223> Identification of the first type of dengue virus oligonucleotide probe <400 >15 ccaggatcgg tggaggcc 18

<210> SEQE) NO: 16 < 211 > 23 < 212 > DNA < 213 > Artificial Sequence < 220 >< 223 > Identification of the first type of dengue virus nucleotide probe < 400 >16 tgcagttatc caagatgagg aaa 23 <210> SEQ ID NO: 17 < 211 > 18 Q < 212 > DNA < 213 > Artificial Sequence < 220 >< 223 > Identification of the second type of dengue virus Glycoside probe <400>17 cttggaatgc tgcaggga 18 <210>SEQ ID NO: 18 <211>22 35 200927939 <212>DNA <213 > Artificial sequence <220><223> Identification Nucleotide probe for the second type of dengue virus <400>18 ttgtcctctt ctcaggcaga at 22

<210>SEQE)NO: 19 <211>20 <212>DNA<213>Artificial sequence<220><223> Identification of second type dengue virus nucleotide probe <;400>19 caatgagagg agcgaagaga 20 <210> SEQ ID NO: 20 <211>18 <212>DNA G < 213 > Artificial Sequence <220><223> Identification of Type 2 Dengue Virus Nucleotide probe <400 >20 gcaatctatg gggctgcc 18 <210> SEQ ID NO: 21 <211>16 <212> DNA 36 200927939 <213 > artificial sequence. <220>< 223 > The nucleotide probe for identifying the second type of dengue virus <400 >21 ggtggtgact gaggac 16 <210>SEQE)NO: 22 <211>19 <212>DNA <213 > artificial sequence <220>< 223 > Identification of a second type of dengue virus nucleotide probe <400 >22 ttctggaaaa ctcataaca 19 <210> SEQEDNO: 23 <211>22 <212>DNA <;213>Artificial sequence 〇<220> <223> Identification of a second type of dengue virus nucleotide probe <400 >23 ctggacatga gtgggttacg ga 22 <; 210 > SEQ ID NO: 24 < 211 > 22 < 212 > DNA < 213 > Artificial Sequence 37 200927939 < 220 >< 223 > Identification of the second type of dengue virus nucleotide probe < 400 >24 ccactctagt gcagcacaaa ga 22 <210> SEQ ID NO: 25 < 211 > 22 < 212 > DNA < 213 > Artificial Sequence Ο < 220 >< 223 > Identification of nucleosides of the third type of dengue virus Acid probe <400 >25 agacatcgct ctgtctcatg at 22 <210>SEQ ID NO.26 <211>21 <212>DNA<213> Artificial sequence 〇<220><223> The nucleotide probe for the third type of dengue virus <400 >26 atgttaccag caacacttgc t 21 <210>SEQ ID NO.·27 - <211>23 <212>DNA<213> 38 200927939 <220><223> oligonucleotide probe for identifying type III dengue virus <400 >27 gcttgaatac ctttgtgttg aag 23 <210>SEQE)NO: 28 <211>20 <212>DNA<213> artificial sequence

<220><223> Identification of a nucleotide probe for the third type of dengue virus <400 >28 agtggtgacc aagaaggagg 20 <210> SEQ ID NO: 29 <211>20 <212>DNA <213> Artificial sequence Ο <220 ><223> Identification of oligonucleotide probe of type III dengue virus <400 >29 catcacagaa aactgtggga 20 <210> SEQ ID NO: 30 <211>22 <;212>DNA<213>Artificial sequence 39 200927939 <220><223> Identification of nucleotide probe for the third type of dengue virus <400 >30 agtgtcaggg aagttgatac ac 22 <210> SEQIDNO : 31 <211>21 <212>DNA <213> Artificial sequence ❹ <220>< 223 > Identification of a third type of dengue virus nucleotide probe <400>31 acgctccaat tcaagatgaa g 21 <210> SEQ ID NO: 32 < 211 > 25 < 212 > DNA < 213 > Artificial Sequence 〇 < 220 >< 223 > Identification of the third type of dengue virus nucleotide probe < 400 >32 caatgaatgg attaccgact ttgtt 25 <210>SEQIDNO:33 <211>26 <212>DNA <213 > artificial sequence 40 200927939 <220><223> Identification of a nucleotide probe for the fourth type of dengue virus <400 >33 gtcaacaatg acattgctgt gtttga 26 <210> SEQ ID NO: 34 <211>25 <212> DNA <213 > artificial sequence

<220><223> Identification of a nucleotide probe for the fourth type of dengue virus <400 >34 gatactgatt ggattcagga aggag 25 <210> SEQ ID NO: 35 <211>23 <212>DNA <213> Artificial sequence © <220><223> Identification of oligonucleotide probe of type 4 dengue virus <400 >35 ctgagaatac caacagtgtg acc 23 <210> SEQ ID NO: 36 * <211&gt ; 23 <212>DNA <213> Artificial sequence 41 200927939 <220><223> Identification of nucleotide probes for type 4 dengue virus <400 > 36 ♦ aggaattact ctgtttctgg gtt 23 <;210>SEQEDNO: 37 <211>19 <212>DNA <213 > Artificial Sequence <220>

<223> Identification of nucleotide probes for type 4 dengue virus <400 >37 ctttggagaa tgccccgga 19 <210> SEQ ID NO: 38 <211>25 <212>DNA <213><220><223> Identification of oligonucleotide probe of type 4 dengue virus 〇 <400>38 cacaattcag gaggattgtg accat 25 <210> SEQ ID NO: 39 <211>22 <212>DNA <213 > artificial sequence 42 <220> 200927939 < 223 > Identification of a nucleotide probe for the fourth type of dengue virus. <400 >39 cagcccaata gaagacatcg ag 22 <210> SEQ ID NO: 40 <211>22 <212>DNA <213 > artificial sequence

<220><223> Identification of a nucleotide probe for the fourth type of dengue virus <400 >40 ggccaatttt agagctggga ga 22 <210> SEQ ID NO: 41 <211>652 <212> DNA <213 > Dengue 1 virus < 220 > capsid sequence <223> Identification of the characteristic region of the first type of dengue virus <400 > 41 1 aatatgctga aacgcgcgag aaaccgcgtg tcaactgttt cacagttggc 51 gaagagattc tcaaaaggat tgctctcagg ccaaggaccc atgaaattgg 101 tgatggcttt catagcattc ctaagatttc tagccatacc cccaacagca 151 ggaattttgg ctagatgggg ctcattcaag aagaatggag cgatcaaagt 201 gttacggggt ttcaagaaag aaatctcaaa catgttgaat ataatgaata 251 gaaggaaaag atctgtgacc atgctcctta tgctgctgcc cacagccttg 301 gcgttccatt tgaccacacg agggggagag ccgcacatga tagttagcaa 351 gcaggaaaga ggaaagtcac ttttgtttaa gacctctgca ggtgtcaaca 43 200927939 401 tgtgcaccct tattgcgatg gatttgggag agttatgtga ggacacaatg 451 acttacaaat gccctcgaat cactgaggcg gaaccagatg acgttgattg 501 ttggtgcaat gccacagaca Catgggtgac ctatggaaca tgttctcaaa 551 ctggcgagca ccgacgagac a Aacgttccg tcgcactggc cccacacgtg 601 ggacttggtc tagaaacaag aaccgaaacg tggatgtcct ctgaaggcgc 651 tt <210>SEQE)NO: 42 <211 >777 <212>DNA <213 > Dengue 1 virus

< 220 > envelope sequence <223 > Identification of the characteristic region of the first type of dengue virus <400 > 42 1 atcacaagaa ggagcaatgc acactgcgtt gactggagcg acagaaatcc 51 aaacgtctgg aacgacaaca atttttgcag gacacttgaa atgtagacta 101 aaaatggaca aactgactct aaaagggatg tcatatgtga tgtgcacagg 151 ctcattcaag ctagagaagg aagtggctga gacccagcat ggaactgttc 201 tagtgcaggt taaatacgaa ggaacagatg caccatgcaa gatccctttt 251 tcgacccaag atgagaaagg agtaacccag aatgggagat tgataacagc 301 caaccccata gtcactgaca aagaaaaacc agtcaacatt gaggcagaac 351 caccttttgg tgagagttac atcgtggtag gagcaggtga aaaagctttg 401 aaactaagct ggttcaagaa aggaagcagc atagggaaaa tgtttgaggc 451 aactgcccga ggagcacgaa ggatggccat actgggagac accgcatggg 501 actttggttc tataggagga gtgttcacgt ctgtgggaaa attagtacac 551 cagatttttg gaactgcata tggagttttg ttcagcggtg tttcttggac 601 catgaaaata ggaatagggg ttctgctgac atggctagga ttaaactcaa 651 ggagcacgtc cctttcgatg acgtgcattg cagttggcct ggtcacactg 701 751 tacctaggag aggcagagaa tcatggttca ctcaaatgtg ggcggat Tcg gaagtgg ggatgtgtaa ttaattggaa <210> SEQ ID NO: 43 <211>419 44 200927939 <212>DNA <213 > Dengue 1 virus <220 > non-structure protein 1 sequence <223> Identification of the first type characterized in dengue virus of region < 400 > 43 1 gacatggggt actggataga aagtgaaaag aacgagacct ggaagctggc aagagcctcc ttcatagaag ttaaaacatg cgtctggcca aaatcccaca 101 ctctatggag caatggagtt ctggaaagtg aaatgataat tccaaagata 151 tatggaggac caatatctca gcacaactac agaccaggat atttcacaca 201 aacagcaggg ccgtggcacc taggcaagtt ggaactagat tttgatttgt 251 gtgagggtac cacagttgtt gtggatgaac attgtggaaa tcgaggacca 51 301 tctcttagaa Ccacaacagt cacaggaaag ataatccatg aatggtgttg 351 cagatcttgt acgctaccac ccttacgttt caaaggagaa gatgggtgtt 401 ggtacggtat ggaaatcag <210>SEQIDNO: 44 <211 >637 <212>DNA < 213 > Dengue 1 virus < 220 > non-structure protein 3 sequence <223> Identification of the characteristic area of the first type of dengue virus <4〇〇>44 1 tcatggatga agcacatttc accgatccat ccagca tagc ggccagaggg 51 tacatctcaa cccgagtggg tatgggtgaa gcagctgcga tcttcatgac 101 agccactccc ccaggatcgg tggaggcctt tccacagagc aatgcagtta 151 tccaagatga attcctgaga ggaaagagac aaacttccga gccgacaggg taatagaccc gatcatggaa ctcaggctat 201 gactggatca ctgacttccc aggtaaaaca gtctggtttg ttccaagcat 251 caaatcagga aatgacattg ccaactgttt aagaaagaat gggaaacggg 301 tgattcaatt gagcaggaaa acctttgaca cagagtacca aaaaacaaaa 351 aacaacgact gggactatgt cgtcacaaca gacatctccg aaatgggagc 401 aagacggtgt ctgaaaccgg 451 taatactaaa agatggtcca gagcgtgtca ttctagcagg accgatgcca 501 gtgactgtgg ccagtgccgc ccagaggaga ggaagaattg gaaggaacca 551 aaataaggaa ggtgatcagt acatctacat gggacagcct ttaaacaatg 45 200927939 601 atgaggatca cgctcattgg acagaagcaa aaatgct < 210 > SEQIDNO: 45 < 211 > 652 < 212 > DNA < 213 > Dengue 2 virus < 220 > capsid sequence <223> Identification of the characteristic region of the second type of dengue virus <400 > 45 1 aatatgctga aacgcgagag aaaccgcgtg tcgactgtgc agcagctgac 51 aaagagattc tca cttggaa tgctgcaggg acgaggacca ttgaaactgt 101 tcatggccct ggtggcgttc cttcgtttcc taacaatccc gccaacagca 151 gggatattga aaagatgggg aacaatcaaa aaatcaaagg ctatcaatgt 201 tttgagaggg ttcaggaaag agattggaag gatgctgaac atcttgaaca 251 ggagacgcag aactgcaggt gtgatcatta tgatgattcc aacagtgatg 301 gcgttccatt taaccacacg taacggagaa ccacacatga tcgtcagtag 351 acaagagaaa gggaaaagtc ttctgtttaa aacagaggat ggtgtgaaca 401 tgtgtaccct catggccatg gaccttggtg aattgtgtga agacacaatc 451 acttataatt gtcctcttct caggcagaat Gaaccagaag acatagactg 501 ttggtgcaac tctacgtcta catgggtaac ttatgggaca tgtaccgcca 551 caggagaaca cagaagggaa aaaagatcag tggcactcgt tccacatgtg 601 651 ggaatgggac ct tggagacacg aactgaaaca tggatgtcat cagaaggggc 〇

<210>SEQ ID NO: 46 < 211 > 777 < 212 > DNA < 213 > Dengue 2 virus < 220 > envelope sequence < 223 > Identification of characteristic regions of the second type of dengue virus < 400 > 46 1 1 atcccaagaa ggggctatgc atacagcact cacaggggcc acggaaatcc 46 200927939 51 agatgtcatc aggaaactta ctgttcacag gacatcttaa gtgcaggctg 101 agaatggaca aactacagct caaaggaatg tcatattcta tgtgtacagg • 151 aaagtttaaa gttgtgaagg aaatagcaga aacacaacat ggaacaatag 201 ttatcagagt acaatatgaa ggggacggtt ctccgtgtaa gatccctttt 4 251 gagataatgg atttggaaaa aagacatgtc ttaggtcgct tgattacagt 301 caacccaatt gttacagaaa aagatagccc agtcaacata gaagcagaac 351 ctccattcgg agacagctac atcattatag gagtagagcc gggacaactg 401 aagctcagct ggtttaagaa aggaagttct attggccaaa tgtttgagac 451 aacaatgaga ggagcgaaga gaatggccat tttaggtgac acagcttggg 501 attttggatc cctgggagga gtgtttacat ctataggaaa ggctctccac 551 caagtctttg gagcaatcta tggggctgcc ttcagtgggg tttcatggac 601 tatgaaaatc ctcataggag tcgtcatcac atggatagga atgaattcac 6 51 gcagcacctc actgtctgtg tcactagtat tggtgggggt cgtgacactg o 701 tatttgggag tcatggtgca ggccgatagt ggttgcgttg tgagttggaa 751 aaacaaagaa ctgaaatgtg gtagtgg <210>SEQIDNO:47 <211 >419 <212>DNA < 213 > Dengue 2 virus < 220 > non- Structure protein 1 sequence <223> identifies the characteristic area of the second type of dengue virus <400 >47

gaaagcctct ttcattgaag ttaaaagctg ccactggcca aagtcacaca 101 ctctctggag taatggagtg ctagaaagtg agatgataat tccaaagaat 151 tttgctggac cagtgtcaca acacaactac agaccaggct atcatacaca 201 aacggcagga ccctggcatc taggtaagct tgagatggac tttgatttct 251 gcgaaggaac cacagtggtg gtgactgagg actgtggaaa tagaggaccc 301 tctttaagaa caactactgc ttctggaaaa ctcataacag aatggtgctg 351 ccgatcttgc acattaccac cgctaaggta cagaggtgag gatggatgct 401 ggtatgggat 1 gatatgggtt attggataga aagtgcactt aatgacacat ggaagattga 51 Ggaaatcag 47 200927939 <210> SEQ ID NO: 48 < 211 > 637 < 212 > DNA < 213 > Dengue 2 virus < 220 > non-structure protein 3 sequence < 223 > Identification of the second type of dengue virus the feature region < 400 > 48 1 tcatggatga agcccatttc acagacccag caagtatagc agctagagga 51 tacatttcaa ctcgagttga gatgggtgaa gcagctggga ttttcatgac 101 agctactcct cctggaagca gagacccatt tcctcagagc aatgcaccaa 151 tcatggatga agaaagggaa atccctgagc gttcgtggaa ttctggacat 201 gagtgggtta cggattttaa agggaagact gttt ggtttg ttccaagtat 251 aaaagcagga aatgatatag cagcttgcct gagaaagaat ggaaagaaag 301 tgatacaact cagtaggaag acttttgatt ctgagtatgt caagactagg 351 accaatgatt gggactttgt ggtcacgact gacatttcag aaatgggtgc 401 taacttcaag gctgagaggg ttatagaccc caggcgctgc atgaaaccag 451 tcatactaac tcctggcagg agatggtgaa gagcgggtga acatatacat aaagaagcta gggggaacct acccatgcca 501 gtgacccact ctagtgcagc acaaagaaga gggagagtag gaagaaatcc 551 601 aaaaaatgaa atgaagactg aatgaccagt tgcacactgg agatgct ctggaaaatg

<210> SEQ ID NO: 49 Ο <211>652 <212>DNA < 213 > Dengue 3 virus < 220 > capsid sequence <223> Identification of the characteristic region of the third type of dengue virus, < 400 > 49 1 aatatgctga aacgcgtgag aaaccgtgtg tcaactggat cacagttggc, 51 gaagagattc tcaagaggat tgctgaacgg ccaaggacca atgaaattgg 101 ttatggcgtt catagctttc ctcagatttc tagccattcc accaacagca 151 ggagtcttgg ctagatgggg aaccttcaag aagtcggggg ctattaaggt 201 cctnaaaggc ttcaagaagg agatctcaaa catgctgagc attatcaaca 48 200927939 251 aacggaaaaa gacatcgctc tgtctcatga tgatgttacc agcaacactt 301 gctttccact tgacttcacg agatggagag ccgcgcatga ttgtggggaa 351 gaatgaaaga gggaaatccc tactttttaa gacagcctct ggaatcaaca 401 tgtgcacact catagccatg gatttgggag agatgtgtga tgacacggtc 451 acttacaaat gccccctcat taccgaagtg gaacctgaag acattgactg 501 ctggtgcaac cttacatcga catgggtgac ttacggaacg tgcaatcaag 551 ctggagagca tagacgcgac aagagatcag tggcgttagc tccccatgtc 601 ggcatgggac tggacacacg cacccaaacc tggatgtcgg ctgaaggagc 651 tt

<210> SEQ ID NO: 50 < 211 > 777 < 212 > DNA < 213 > Dengue 3 virus < 220 > envelope sequence < 223 > Identification of the characteristic region of the third type of dengue virus < 400 > 50 1 atcgcaagag ggagcaatgc acacagcact gacaggagct acagagatcc 51 aaaactcagg aggcacaagc atttttgcgg ggcacttgaa atgtagactc 101 aagatggaca aattggaact caaggggatg agctatgcaa tgtgcttgaa 151 tacctttgtg ttgaagaaag aagtctccga aacgcagcat gggacaatac 201 tcattaaggt tgagtacaaa ggggaagatg caccttgcaa gattcctttc 251 tccacagagg atggacaagg gaaagcccac aatggcagac tgatcacagc 301 caacccagtg gtgaccaaga aggaggagcc tgtcaatatt gaggctgaac 351 ctccttttgg ggaaagtaac atagtgattg gaattggaga caaagccttg 401 aaaatcaact ggtacaagaa gggaagctcg attgggaaga tgttcgaggc 451 cactgccaga ggtgcaaggc gcatggccat cttgggagac acagcctggg 501 actttggatc agtgggtggt gttctaaatt cattagggaa aatggtgcac 551 caaatatttg gaagtgctta cacagccctg tttagtggag tctcntggat 601 aatgaaaatt ggaataggtg tcctcttaac ctggataggg ttgaattcaa 651 aaaacacttc catgtcattt tcatgcat Tg tgataggaat cattacactc 701 751 tatctgggag aggaaaagaa ccgtggtaca ctcaaatgtg agctgacatg gaagtgg 999tgtgtca taaactggaa 49 200927939 <210> SEQ ID NO: 51 <211>419 <212>DNA < 213 > Dengue 3 virus < 220 > non-structure protein 1 sequence < 223 > identification of a third type wherein dengue viruses region < 400 > 51 1 gacatgggct attggataga aagccaaaag aatggaagtt ggaagctaga 51 aaaagcatcc ctcatagagg tgaaaacctg cacatggcca aaatcacaca 101 ctctttggag caatggtgtg ctagagagtg acatgatcat cccaaagagt 151 ctggctggtc ctatttcgca acacaaccac aggcccggat accacaccca 201 aacggcagga ccctggcact taggaaaatt ggagctggac Ttcaactatt 251 gtgaaggaac aacagttgtc atcacagaaa actgtgggac aagaggccca 301 tcattgagaa caacaacagt gtcagggaag ttgatacacg aatggtgttg 351 401 ccgctcgtgc ggtatggcat acacttcctc ggaaattag ccttgcgata catgggagag gacggctgct

<210> SEQ ID NO: 52 < 211 > 637 Q < 212 > DNA < 213 > Dengue 3 virus < 220 > non-structure protein 3 sequence < 223 > Identification of the characteristics of the third type of dengue virus region < 400 > 52 1 taatggatga ggcccatttc acagacccag ccagtatagc ggctagaggg n 51 tacatatcaa ctcgtgtagg aatgggagag gcagccgcaa ttttcatgac, 101 agcaacaccc cctggaacag ctgatgcctt tcctcagagc aacgctccaa 151 ttcaagatga agaaagggac atnccagaac gctcatggaa ttcaggcaat 201 gaatggatta ccgactttgt tgggaagacg gtgtggtttg tccctagcat 251 caaagccgga aatgacatag caaactgctt gcgaaaaaat ggaaaaaagg 50 200927939 301 tcattcaact tagtaggaag acttttgaca cagaatatca aaagaccaaa 351 ctgaatgatt gggacttcgt ggtgacaaca gacatttcag aaatgggagc 401 caatttcaaa gcagacagag tgatcgaccc aagaagatgt ctcaagccag 451 tgattttgac agatggaccc gagcgngtga tcctggcngg accaatgcca 50X gtcaccgcag cgagcgctgc gcaaaggaga gggagagttg gcaggaaccc 551 acaaaaagaa aatgaccagt acatattcac gggccagccc ctcaacaatg 601 atgaagacca tgctcactgg acagaagcaa aaatgct < 210 > SEQ ID NO: 53 <211>652

<212>DNA < 213 > Dengue 4 virus < 220 > capsid sequence <223 > Identification of characteristic region of type 4 dengue virus <400 > 53 1 aatatgctga aacgcgagag aaaccgcgta tcaacccctc aagggttggt 51 gaagagattc tcaaccggac ttttttccgg gaaaggaccc ttacggatgg 101 tgctagcatt catcacgttt ttgcgagtcc tttccatccc accaacagca 151 gggattctga aaagatgggg acagttgaag aaaaataagg ccatcaagat 201 actgattgga ttcaggaagg agataggtcg catgctgaac atcttgaatg 251 ggagaaaaag gtcaacaatg acattgctgt gtttgattcc caccgtaatg 301 gcgtttcact tgtcaacaag agatggcgaa cccctcatga tagtggcaaa 351 acacgaaagg gggagacctc tcttgtttaa gacaacagaa ggaatcaaca 401 aatgcactct tattgccatg gacctgggtg aaatgtgtga ggacactgtc 451 acgtataaat gccctctact ggtcaatacc gaacctgaag Cat 。。。。。。。。。。。。。。。。 A < 213 > Dengue 4 virus < 220 > envelope sequence < 223 > Identification of the characteristic area of the fourth type of dengue virus <400 > 54 1 atctcaggaa ggagccatgc attctgccct cgccggagcc acagaagtgg 51 attccggtga tggaaatcac atgtttgcag gacatctcaa gtgcaaagtt 101 cgtatggaga aattgagaat taagggaatg tgtgctcagg 151 aaagttctca attgacaaag agatggcaga aacacagcat gggacaacag 201 tggtgaaagt caagtatgaa ggtgctggag ctccgtgtaa agtccccata 251 gagataagag atgtgaacaa ggaaaaagtg gttgggcgca tcatctcatc 301 cacccctttt gctgagaata ccaacagtgt gaccaacata gaattggaac 351 ccccttttgg ggacagctac atagtgatag gtgttggaga cagtgcatta 401 acactccatt ggttcaggaa agggagctcc attggcaaga tgtttgagtc 451 cacatacaga ggtgcaaaac gaatggccat tctaggtgaa acagcttggg 501 attttggttc tgttggtgga ctgttcacat tcatacacga Cattgggaaa ggctgtacac 551 caggtttttg gaagtgtgta tacaaccatg tttggaggag tctcatggat 601 ggttagaatc ctaattgggt tcttagtgtt gtggattggc acgaattcaa 651 gaaacacttc aatggcaatg acgtgcatag ctgttggagg aattactctg 701 751 tttctgggtt tgggaaa Gaa tcacagttca agcagacatg ttaaaatgtg gaagcgg ggttgtgtgg tgtcatggag 〇〇<210> SEQIDNO: 55 <211>419 <212>DNA < 213 > Dengue 4 vims < 220 > non-structure protein 1 sequence » <223> identifying a fourth type feature region dengue virus, < 400 > 55 1 gacatgggtt attggataga gagctcaaaa aaccagacct ggcagataga 51 gaaagcatcc cttattgagg tgaaaacatg tctgtggccc aagacccaca 101 cgttgtggag caatggagtg ctggaaagcc agatgctcat tccaaaatca 52 200927939 151 tatgcaggcc ctttttcaca gcacaattac cgccagggct atgccacgca 201 aaccgtgggc ccatggcact taggcaaatt ggagatagac tttggagaat 251 gccccggaac aacagtcaca attcaggagg attgtgacca tagaggccca 301 tctttgagga ccaccactgc atctggaaaa ctggtcacgc aatggtgctg 351 ccgctcctgc acgatgcctc ccttaaggtt cttgggagaa gatggatgtt 401 ggtatgggat ggagattag < 210 > SEQIDNO: 56 < 211 > 637 < 212 > DNA < 213 > Dengue 4 virus < 220 > non-structure protein 3 sequence <223> identifies the characteristic area of the fourth type of dengue virus <400 &gt ;56

1 tgatggatga agcacatttc acngaccctt ctagtgtcgc ggctagagga 51 tacatctcaa ccagggtgga aatgggagag gcagcagcta tcttcatgac 101 tgcaacccct cctggaacga cagatccctt cccccagagc aacagcccaa 151 tagaagacat cgagagggaa atcccagaaa ggtcatggaa cacagggttc 201 gactggataa cagactacca agggaaaact gtgtggtttg ttcccagcat 251 aaaagctgga aatgacattg caaattgttt gagaaagtcg ggaaagaaag 301 tgatccagtt gagcaggaaa acctttgaca cagagtatcc aaaaacgaaa 351 ctcacggact gggattttgt ggtcaccaca gacatatctg aaatgggggc 401 caattttaga Gctgggagag tgatagaccc caggagatgc ctcaagccag 451 ttatcctaac agatgggcca gagagagtta ttttagcagg tcccattcca 501 gtgactccag caagcgctgc tcagagaaga gggcgaatag gtaggaatcc 551 601 agcacaagaa atgaagatca gatgaccaat tgcccactgg atgttttctc acagaagcaa tggagaccca aaatgct ctaaagaatg 53