1293090 玫、發明說明： 【發明所屬之技術領威】 本發明係有關於設計PCR(聚合酶鍵反應）引子之裝置及方 法。更特別地，本發明係有關於用於敏感性PCR债測冠狀病毒 之組合物，上述冠狀病毒係嚴重急性呼吸道症候群（severeacute respiratory syndrome ’簡稱SARS)之致病原。本發明之裝置與方 法亦可用於設計偵測其他RNA或DNA病原的組合物。 【先前技術】 PCR，也就是所謂的聚合酶鏈反應’是目前生物學與生醫 學實驗室常用的操作方法之一。PCR的優點是可將少量核酸增 幅為百萬至十億倍的量。此種方法對分子層級的基因學分析開 啟 了一個新紀元（Glennon，M· and Cormican，M_ (2001) Detection and diagnosis of mycobacterial pathogens using PCR. Expert. Rev. Mol. Diagn., 1, 163-174; Jain? K. K. (2002) Current trends in molecular diagnostics. Med. Device Technol., 13, 14-18)。更確切地，PCR可以應用作為研究與診斷的工具，例如 用於偵測病原性病毒或細菌的存在。 雖然PCR省時又敏感，偵測時卻會有偽陽性訊號的嚴重問 題。最常見的瑕疵發生原因為，引子本身發生二聚化，以及引 子對於模板不適當的煉合。 減低瑕疵的一種技術牽涉到採用兩對順向與反向PCR引 子，其中第二對引子結合到第一對引子所辨識並增幅之序列 内。第一對引子或稱「外引子對（outer primer set)」增幅標的序 列所給區域，也就是第一次PCR之「外增幅區（outer amplicon)」。接著，於第二次或稱「巢式（nested)」PCR，第二 1293090 對引子或稱「内引子對（inner primer set)」辨認外增幅區内的序 列，以增幅一較短區域或稱「内增幅區（inner amplicon)」。採 用「巢式引子（nested primers)」可減低增幅錯誤序列的機率，因 為若是第一對引子結合並增幅錯誤序列，第二對引子結合並增 幅同樣錯誤序列的機率將非常低。然而，此技術仍然依賴每對 引子之專一性（參見 http://www.bio.davidson.edu/courses/ genomics/method/NestedPCR.html) 〇 因此，引子設計對於PCR為基的偵測方法實至為重要。引 子之一般要求條件並不複雜（Robertson，J· M· and Walsh-Weller， J. (1998) An introduction to PCR primer design and optimization of amplification reactions. Methods Mol. Biol.9 98, 121-154; Rozen，S. and Skaletsky，H. (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol. Biol., 132, 365-386)，且已知其與某些熱力動力學之相關性，例如，個 別驗基對之配對穩定性可以由最近相鄰模式（Nearest_Neighbor model)計算(SantaLucia, J. Jr.? Allawi, Η. T. and Seneviratne, P. A. (1996) Improved nearest-neighbor parameters for predicting DNA duplex stability. Biochemistry, 35? 3555-3562; Sugimoto, N., Nakano，S·，Yoneyama，M. and Honda，K. (1996) Improved thermodynamic parameters and helix initiation factor to predict stability of DNA duplexes. Nucleic Acids Res·，24，4501-4505) o 但是，為一模板序列選擇好的引子並非易事。不只是計算工程 浩大，最佳化排比機制也非常複雜。因此，電腦輔助引子設計 在生物資訊學也扮演重要角色。 有多種網路為基的服務或離站軟體可進行引子設計，如 PRIDE (Haas，S·，Vingron，M·，Poustka，A. and Wiemann，S· 1293090 (1998) Primer design for large scale sequencing. Nucleic Acids Res” 26，3006-3012)，PRIMER MASTER (Proutski, V. and Holmes, E. C. (1996) Primer Master: a new program for the design and analysis of PCR primers. Comput· Appl. Biosci.， 12, 253-255)，PRIMO (Li，P·，Kupfer，K. C·，Davies，C· J·，Burbee，D·， Evans，G. A. and Garner，H. R· (1997) PRIMO: A primer design program that applies base quality statistics for automated large-scale DNA sequencing. Genomics, 40, 476-485) ^ PrimeArray (Raddatz，G·，Dehio，M·，Meyer，T. F. and Dehio，C. (2001) PrimeArray: genome-scale primer design for DNA-microarray construction. Bioinformatics., 17, 98-99) J Primer3 (Rozen, S. and Skaletsky, H. (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol. Biol., 1325 365-386) j Prime (Eberhardt, N. L. (1992) A shell program for the design of PCR primers using genetics computer group (GCG) software (7.1) on VAX/VMS systems· 13，914-917)，以及 Web1293090 玫,发明说明: [Technical Leadership of the Invention] The present invention relates to an apparatus and method for designing a PCR (polymerase bond reaction) primer. More particularly, the present invention relates to a composition for a sensitive PCR debt detecting coronavirus, the causative agent of the above-mentioned coronavirus system severe respiratory respiratory syndrome (SARS). The devices and methods of the present invention can also be used to design compositions for detecting other RNA or DNA pathogens. [Prior Art] PCR, also known as polymerase chain reaction, is one of the commonly used methods of operation in biological and biomedical laboratories. The advantage of PCR is that a small amount of nucleic acid can be increased by a million to a billion times. This approach opens a new era in the genetic analysis of molecular levels (Glennon, M. and Cormican, M_ (2001) Detection and diagnosis of mycobacterial pathogens using PCR. Expert. Rev. Mol. Diagn., 1, 163-174; Jain? KK (2002) Current trends in molecular diagnostics. Med. Device Technol., 13, 14-18). More specifically, PCR can be applied as a tool for research and diagnosis, for example, to detect the presence of a pathogenic virus or bacteria. Although PCR is time-sensitive and sensitive, there are serious problems with false positive signals when detecting. The most common cause of cockroaches is the dimerization of the primer itself and the improper smelting of the template for the template. One technique for reducing enthalpy involves the use of two pairs of forward and reverse PCR primers, wherein the second pair of primers is incorporated into the sequence identified and amplified by the first pair of primers. The first pair of primers, or the region given by the "outer primer set" increment, is the "outer amplicon" of the first PCR. Then, in the second or "nested" PCR, the second 1293090 identifies the sequence in the outer amplification region for the primer or the "inner primer set" to increase the length of a shorter region or "Inner amplicon". The use of "nested primers" can reduce the probability of an amplitude-increasing sequence, because if the first pair of primers combines and amplifies the wrong sequence, the probability of the second pair of primers combining and augmenting the same error sequence will be very low. However, this technique still relies on the specificity of each pair of primers (see http://www.bio.davidson.edu/courses/genomics/method/NestedPCR.html). Therefore, the primer design is a PCR-based detection method. It is important. The general requirements for the introduction are not complicated (Robertson, J. M. and Walsh-Weller, J. (1998) An introduction to PCR primer design and optimization of amplification reactions. Methods Mol. Biol. 9 98, 121-154; Rozen , S. and Skaletsky, H. (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol. Biol., 132, 365-386), and its correlation with certain thermodynamics is known, For example, the pairing stability of individual test pairs can be calculated from the nearest neighbor model (SantaLucia, J. Jr.? Allawi, Η. T. and Seneviratne, PA (1996) Improved nearest-neighbor parameters for predicting DNA Diverse stability. Biochemistry, 35? 3555-3562; Sugimoto, N., Nakano, S., Yoneyama, M. and Honda, K. (1996) Improved thermodynamic parameters and helix initiation factor to predict stability of DNA duplexes. Nucleic Acids Res ·, 24,4501-4505) o However, choosing a good primer for a template sequence is not an easy task. Not only is the computational engineering vast, but the optimal alignment mechanism is also very complicated. Therefore, computer-aided primer design also plays an important role in bioinformatics. There are a variety of network-based services or off-site software for primer design, such as PRIDE (Haas, S., Vingron, M., Poustka, A. and Wiemann, S. 1293090 (1998) Primer design for large scale sequencing. Nucleic Acids Res" 26,3006-3012), PRIMER MASTER (Proutski, V. and Holmes, EC (1996) Primer Master: a new program for the design and analysis of PCR primers. Comput· Appl. Biosci., 12, 253 -255), PRIMO (Li, P., Kupfer, K. C., Davies, CJ, Burbee, D., Evans, GA and Garner, H. R. (1997) PRIMO: A primer design program that Genomics, 40, 476-485) ^ PrimeArray (Raddatz, G., Dehio, M., Meyer, TF and Dehio, C. (2001) PrimeArray: genome-scale primer Design for DNA-microarray construction. Bioinformatics., 17, 98-99) J Primer3 (Rozen, S. and Skaletsky, H. (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol. Biol., 1325 365-386) j Prime (Eberhardt, N. L (1992) A shell program for the design of PCR primers using genetics computer group (GCG) software (7.1) on VAX/VMS systems· 13,914-917), and Web
Primer (http://genome-www2.stanford.edu/ cgi-bin/SGD/web-primer)。使用者可以定義這些工具之目錄所歹丨J出的參數，接著 擷取標的模板序列的數個引子對。然而，其中大部分只能進行 單一序列檢索。此外，引子煉合條件的計算也常被簡化為溶化 溫度（Tm)的簡單方程式，而無視於引子本身所含序列内容。例 如，也許會有引子二聚化，牵涉到引子-二聚體，如自體-二聚體 及跨引子-二聚體，這些都沒有列入考量。因此，對於評估引子 適用性的方法以及對應的電腦或網路為基的引子設計工具仍有 所需求。 8 1293090 此外，新致病原的出現，與其相關的人員傷亡，更強化對 於正確偵測方法的需求。特別是考慮到冠狀病毒新種，嚴重急 性呼吸道症候群（Severe Acute Respiratory Syndrome，簡稱 SARS) 之潛在致病原。 冠狀病毒於1937年首分離於雞，並在1965年經由培養人 類纖毛胚胎氣管而於體外得到第一個人類冠狀病毒，並發現與 人類感 冒相關 （see http://www-micro.msb.le.ac.uk/3035/Coronaviruses.html) 〇 於2002年末，中國大陸、越南、加拿大，及香港陸續報導 有病因未明且會危及生命的呼吸道疾病。病患表現出發燒，乾 咳，呼吸困難，頭痛，以及由於肺泡損傷引起進行性呼吸衰竭， 並造成血氧過低與死亡（Tsang KW et al_, (2003) J· MM· 348:1975-1983). The syndrome was designated severe acute respiratory syndrome (SARS) and has become a serious global endemic infectious disease in over 30 countries (Drosten CS et al.5 (2003) Engl. J. Med. 348:1967-1976; Ksiazek TG et al·, (2003) N. Engl J. Med. 348:1953-1966; Peiris JS et al.5 (2003) Z⑽cd 361:1767-1772)。全世界大約有4%的SARS病患死亡（參 見 http://www.who.int/csr/sarscountry/ 2003一04_04/en/)。Primer (http://genome-www2.stanford.edu/cgi-bin/SGD/web-primer). The user can define the parameters of the catalog of these tools, and then retrieve several pairs of primers for the target template sequence. However, most of them can only perform a single sequence search. In addition, the calculation of the synthesis conditions of the primer is often simplified to a simple equation of the melting temperature (Tm), regardless of the sequence content contained in the primer itself. For example, there may be primer dimerization involving primer-dimers such as auto-dimers and cross-primers-dimers, which are not considered. Therefore, there is still a need for a method for assessing the applicability of primers and a corresponding computer or network based primer design tool. 8 1293090 In addition, the emergence of new pathogens, associated with casualties, strengthens the need for correct detection methods. In particular, considering the new coronavirus species, the potential pathogen of Severe Acute Respiratory Syndrome (SARS). The coronavirus was first isolated from chicken in 1937, and in 1965 the first human coronavirus was obtained in vitro by culturing human ciliated embryonic trachea and found to be associated with the human cold (see http://www-micro.msb.le .ac.uk/3035/Coronaviruses.html) At the end of 2002, mainland China, Vietnam, Canada, and Hong Kong reported successively respiratory diseases that were unexplained and life-threatening. The patient's performance begins with burning, dry cough, difficulty breathing, headache, and progressive respiratory failure due to alveolar damage, and causes hypoxemia and death (Tsang KW et al_, (2003) J. MM. 348: 1975-1983) The syndrome was designated severe acute respiratory syndrome (SARS) and has become a serious global endemic infectious disease in over 30 countries (Drosten CS et al. 5 (2003) Engl. J. Med. 348:1967-1976; Ksiazek TG et Al., (2003) N. Engl J. Med. 348:1953-1966; Peiris JS et al. 5 (2003) Z(10)cd 361:1767-1772). Approximately 4% of SARS patients worldwide die (see http://www.who.int/csr/sarscountry/ 2003-0404/en/).
SARS的致病原為一新種冠狀病毒，命名為SARS相關冠 狀病毒（SARS_assocaitedcoronavirus，簡稱 SARS-CoV)。最近已 確認新種冠狀病毒之完整基因體序列（Marra MA et al·，（2003) Science 300:1399-1404; Rota PA et al.? (2003) Science 300:1394-1399)。目前數個可取得之SARS-CoV病毒株的基因體 序列顯不’新種病原並不屬於已知的任何冠狀病毒群（Drosten CS et al.? (2003) N. Engl. J. Med. 348:1967-1976; Ksiazek TG 1293090 et al.5 (2003) N. Engl. J. Med. 348:1953-1966; Marra MA et al.5 (2003) Science 300:1399-1404; Rota PA et al.? (2003) Science 300:1394-1399)。SARS-CoV僅與人類冠狀病毒中度相關，且推 測SARS-CoV原為動物病毒，最近才發展為成具備可複製性感 染人類之能力的新種（Ludwig B et al·，（2003) /nierWro/og少 46:71_78)。 冠狀病毒為一大型、含套膜、正股RNA病毒家族，其在 動物宿主細胞之細胞質複製（Sidell S et al·，（1983) J. Wro/· 64:761-776)。冠狀病毒顆粒具有外套膜，其上帶有獨特的棒狀 突出物（’club-shaped’ peplomers)，形成似皇冠之外觀，並因此得 名。套膜帶有糖蛋白，包括棘蛋白（Spike (S) protein)，其可與受 體結合及與細胞融合，形成免疫辨認之主要標的抗原。其他潛 在標的包括基質蛋白（Matrix (M) protein)，核殼蛋白 (nucleocapsid (N) protein)，其與RNA病毒基因體有關，以及 Orflab 聚蛋白（P)。（參見 http://www-micro.msb.le.ac.uk/3035/ Coronaviruses.html) 〇 在缺乏疫苗或有效治療劑的狀況下，流行病學上預防與控 制的關鍵在於，隔離潛在病例與裝備嚴格之檢疫政策。不幸地 是，暴露於SARS之個體及社區的隔離與檢疫並無法避免疾病 蔓延，其原因為缺乏早期偵測SARS的方法，如此更突顯出對 於偵測該病毒之早期敏感性診斷方法的需求。 根據引子設計，以PCR偵測編碼標的蛋白基因，可以提供 具有敏感性與正確性、且用於SARS-CoV感染之診斷測試。上 述偵測方法牽涉RNA病毒基因體之反轉錄反應，以及接著進行 之PCR反應（合稱rt-PCR)°PCR偵測之新方法也揭露於相關暫 時申請案，由 Jyh-Lyh Juang 及 Shih Sheng Jiang 申請於 2003 年 1293090 6月30 ’上述内容併入本案作為參考。敏感性pcR偵測係需要 用以減低偽陽性與偽陰性，特別是當病毒只有低複製數時（例如 10個複製數），且更進一步提供證明不穩定樣品之方法。 因此’針對所有RNA及DNA致病原，特別是SARS-CoV， 亟需設計專一性PCR引子的方法，其對應的電腦或網路為基的 引子設計工具’以及包括引子對及巢式引子組之組合物以偵測 病毒。 因此，本發明之目的之一為提供引子設計之方法，以及利 用上述方法之電腦或網路為基的引子設計工具。本發明之又一 目的係應用上述引子設計工具於SARS-CoV之潛在標的基因， 以及提供可敏感性偵測此種新致病原之引子對的組合物。 【發明内容】 本發明之數個實施型態提供基於熱力動力學（SantaLucia，J· Jr. (1998) A unified view of polymer, dumbbell，and oligonucleotide DNA nearest-neighbor thermodynamics. Proc. #如/· jcarf· Sc/· t/Sj，95, 1460-1465)之方法，以評估引子之適用 性。其他實施型態提供採用上述方法之電腦程式或網路介面的 引子設計服務，如引子設計助理（Primer Design Assistant，簡稱 PDA)。於PDA實施例中，可允許於批次式模式進行多條序列之 比對。 本發明之其他實施例提供包括引子序列之組合物，以作為 引子對或巢式引子組而用於SARS-CoV之敏感性偵測（參見序列 表）。這些包括含有偵測棘蛋白、基質蛋白、核殼蛋白及Orflab 聚蛋白基因之序列的組合物。 11 1293090 熟於此技藝人士將了解，如何應用本發明方法與電腦裝置 於產生_其他重要致病原之組合物。的確，統—引子練合條 件的優點是在實驗設計大型PCR增幅時报有用，如於％孔盤 由嗟菌體株製備探針，或任何其他牽涉以不同引子對於相同 PCR反應時間增幅標的模板之應用。 本發明之其纟目的與優點將經以下之說明祕實施例呈現 而更為顯明。本發明之目的及優點亦將經由後附之申請專利範 圍所特別指明之元件與組合而令人更了解。 以上之一般敘述與之後的詳細說明僅係提供作為例示與說 明，並非用以限制本發明所請發明。 所附帶之圖式係併入並構成說明書之部分，以顯示本發明 之實施例，並與說明同時提供作為本發明原理之解釋。 在進一步說明本發明之前，需了解本發明並非限於所述的 特定實施例。也須了解，此處之用語僅係為說明特定實施例，The causative agent of SARS is a new type of coronavirus, named SARS-associated coronavirus (SARS_assocaitedcoronavirus, SARS-CoV for short). The complete genome sequence of the new species of coronavirus has recently been confirmed (Marra MA et al., (2003) Science 300: 1399-1404; Rota PA et al.? (2003) Science 300: 1394-1399). The current gene sequence of several available SARS-CoV strains does not belong to any known coronavirus population (Drosten CS et al.? (2003) N. Engl. J. Med. 348: 1967-1976; Ksiazek TG 1293090 et al. 5 (2003) N. Engl. J. Med. 348:1953-1966; Marra MA et al. 5 (2003) Science 300:1399-1404; Rota PA et al.? (2003) Science 300: 1394-1399). SARS-CoV is only moderately associated with human coronavirus, and it is speculated that SARS-CoV was originally an animal virus and has recently evolved into a new species with the ability to reproducibly infect humans (Ludwig B et al., (2003) / nierWro/ Og less 46:71_78). Coronavirus is a large, enveloped, positive-stranded RNA virus family that is cytoplasmic in animal host cells (Sidell S et al., (1983) J. Wro/64: 761-776). Coronavirus particles have a mantle with unique rod-shaped protrusions ('club-shaped' peplomers) that form a crown-like appearance and are therefore named. The envelope carries glycoproteins, including Spike (S) protein, which bind to the receptor and fuse with the cell to form the primary target antigen for immunological recognition. Other potential targets include Matrix (M) protein, nucleocapsid (N) protein, which is involved in the RNA viral genome, and Orflab polyprotein (P). (See http://www-micro.msb.le.ac.uk/3035/ Coronaviruses.html) 〇 In the absence of vaccines or effective therapeutics, the key to epidemiological prevention and control is to isolate potential cases Strict quarantine policy with equipment. Unfortunately, the isolation and quarantine of individuals and communities exposed to SARS does not prevent the spread of the disease because of the lack of early detection of SARS, which highlights the need for early detection of the virus. According to the design of the primer, PCR detection of the encoded protein gene can provide sensitivity and correctness, and can be used for diagnostic tests of SARS-CoV infection. The above detection method involves the reverse transcription reaction of the RNA virus genome, and the subsequent PCR reaction (collectively called rt-PCR). The new method of PCR detection is also disclosed in the relevant provisional application by Jyh-Lyh Juang and Shih Sheng. Jiang applied for 2003 1293090 June 30 'The above is incorporated into this case for reference. Sensitive pcR detection systems are needed to reduce false positives and false negatives, especially when the virus has only low copy numbers (eg, 10 replicates), and further provide a means of demonstrating unstable samples. Therefore, 'for all RNA and DNA pathogens, especially SARS-CoV, there is no need to design a specific PCR primer, the corresponding computer or network-based primer design tool' and including primer pairs and nested primers. The composition is used to detect a virus. Accordingly, one of the objects of the present invention is to provide a method of designing a primer, and a computer or network based primer design tool using the above method. Still another object of the present invention is to apply the above-described primer design tool to the potential target gene of SARS-CoV, and to provide a composition that can sensitively detect such a new pathogen. SUMMARY OF THE INVENTION Several embodiments of the present invention provide thermodynamics based (Santa Lucia, J. Jr. (1998) A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc. #如/· jcarf · Sc/· t/Sj, 95, 1460-1465) method to evaluate the applicability of the primer. Other implementations provide a primer design service for a computer program or a web interface using the above methods, such as a Primer Design Assistant (PDA). In the PDA embodiment, multiple sequences can be aligned in batch mode. Other embodiments of the present invention provide compositions comprising primer sequences for use as primer pairs or nested primer sets for sensitivity detection of SARS-CoV (see Sequence Listing). These include compositions containing sequences that detect the spine protein, matrix protein, nucleocapsid protein, and Orflab polyprotein genes. 11 1293090 Those skilled in the art will appreciate how to apply the methods and computer devices of the present invention to produce compositions of other important pathogens. Indeed, the advantage of the integration-introduction condition is that it is useful in the experimental design of large-scale PCR amplification, such as the preparation of probes from the sputum strains in the % well plate, or any other template involving different primers for the same PCR reaction time increase. Application. The objects and advantages of the present invention will become more apparent from the following description. The objects and advantages of the invention will be more apparent from the elements and combinations particularly pointed out in the appended claims. The above general description and the following detailed description are merely illustrative and not restrictive of the invention. The accompanying drawings are incorporated in and constitute in the claims Before the invention is further described, it is to be understood that the invention is not limited to the specific embodiments described. It should also be understood that the phrase used herein is for the purpose of describing particular embodiments.
而非用以限制本發明，本發明之範疇僅受限於後附之申請專利 範圍。 W 本說明書提供某範圍内之數值，上述範圍内之數值亦包括 在本發明之範疇之内。這些小範圍之上限與下限可獨立地包括 在該小範圍内，且所述範圍内任何特定排除之限定亦包括在本 發明之範疇内。當所述範圍包括其一或二者之限定時，排除任 一或兩者所包括之範圍也包括在本發明之範_内。 除非另加定義，此處所述之所有技術與科學用語係與本發 明所屬技術領域之人士所熟知者含意相同。雖然與此處所述相 同或荨同之方法與材料也可以用以實施或測試本發明，以下仍 將說明某些方法與材料。所有此處所述之出版品係併入本發明 作為參考資料以揭示與說明相關之方法及/或材料。 12 1293090 須注意的是，除非特別指明，此處所述與後附之申請專利 範圍中，單數形式「一」、「或」、及「該」皆包括所指對象 之複數形式。 此外，除非特別指明，本說明書與申請專利範圍中所有表 現成分含量、反應條件、純度百分比、引子長度等之數字皆含 括「約」字。因此，除非特別定義，本說明書與申請專利範圍 所列數字參數皆為概略值，且依本發明所欲達到之特性而異。 儘管數字範圍與參數所定義出本發明之最廣泛範圍為概略 值，本發明之特定實施例中所列舉之數值仍盡量精確表示。然 而，任何數值皆包括某些在量測上標準差中可能的誤差。 此處所提供之出版品僅係因其早於本案申請日發表而提 供。並非代表承認本發明非早於該出版品之發明。此外，所提 供之出版日期也可能不同於實際出版日期，需個別認定。 【實施方式】 於本發明之某些實施型態，本發明提供基於熱力動力學之 方法，以評估引子之適用性。於某些其他實施型態，允許上述 方法之電腦輔助工具，如PDA。 引子選擇之基本準則包括以下： 1.熔點 引子煉合溫度（Ta)係由熔點（Tm)決定。於一引子對中之可接 受的Tm值差異為5°C内。一引子之Tm值係基於數個物化因素。 引子之Tm值係以如下方程式計算得（Freier，S· M·，Kierzek，R·， Jaeger, J. A·，Sugimoto，N·，Caruthers, Μ. H·，Neilson，T. and Turner，D. H. (1986) Improved free-energy parameters for 13 1293090 predictions of RNA duplex stability. Proc. Natl. Acad. Sci. USA, 83, 9373-9377):Rather than limiting the invention, the scope of the invention is limited only by the scope of the appended claims. W This specification provides numerical values within a range, and values within the above ranges are also included in the scope of the present invention. The upper and lower limits of these sub-ranges can be independently included in the sub-range, and any particular exclusions within the scope are also included in the scope of the invention. When the stated range includes one or both of the definitions, the exclusion of either or both is also included in the scope of the invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, certain methods and materials are described below. All publications described herein are hereby incorporated by reference in their entirety to the extent of the disclosure of the disclosure of the disclosure. 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 In addition, unless otherwise specified, all numerical values, reaction conditions, percent purity, length of primers, etc. in this specification and the scope of the patent application include the word "about". Therefore, unless otherwise specified, the numerical parameters set forth in the specification and claims are intended to be a The numerical values set forth in the particular embodiments of the invention are in the However, any value includes some of the possible errors in the standard deviation of the measurement. The publications provided here are provided only as they are published earlier than the filing date of the case. It is not an admission that the invention is not prior to the invention of the publication. In addition, the date of publication provided may also differ from the actual publication date and is subject to individual identification. [Embodiment] In some embodiments of the invention, the present invention provides a thermodynamic based method for assessing the applicability of a primer. In some other implementations, computer aids such as PDAs permitting the above methods. The basic criteria for primer selection include the following: 1. Melting point The melting temperature (Ta) of the primer is determined by the melting point (Tm). The acceptable Tm value in a pair of primers is within 5 °C. The Tm value of an primer is based on several physical factors. The Tm value of the primer is calculated by the following equation (Freier, S. M., Kierzek, R., Jaeger, J. A., Sugimoto, N., Caruthers, Μ. H., Neilson, T. and Turner, DH). (1986) Improved free-energy parameters for 13 1293090 predictions of RNA duplex stability. Proc. Natl. Acad. Sci. USA, 83, 9373-9377):
Tm (°C) = 59.9 + (0.41xGC 含量）-(675 /引子長度） 於PDA實施例中，例如，提供一預設值，其中順向與反向 引子之GC含量範圍由30%至70%。排除Tm值小於50 °C之引 子。 2-序列複雜性 複雜性低的引子會減低有關序列含量之差別性能量。因 此，不論在順向及反向引子中皆須避免單一核苷酸之重複。也 須避免如‘ATATAT’之連續雙核苷酸重複。 於PDA實施例中，例如，在順向及反向引子皆須排除任何 四或更多的重複（或連續）核苷酸，如AAAA，TTTT，CCCC， 或GGGG。也須避免如‘ATATAT’之連續雙核苷酸重複。 3_ 3’端 C/G 鉗 在雙螺旋DNA股中，核苷酸殘基‘C’或‘G’形成強的配對結 構。引子-模板複合物中具有穩定3’端可提高聚合酶效能（Buck， G· A·，Fox，J. W·，Gunthorpe，M.，Hager，Κ· M·，Naeve，C. W·， Pon，R. T·，Adams，Ρ· S. and Rush，J. (1999) Design strategies and performance of custom DNA sequencing primers. Biotechniques, 27, 528-536)。因此，所選擇之順向或反向引子傾向於3’端具有 C或G 〇 於PDA實施例中，例如，預設3,端C/G鉗以限制所選順 向引子與反向引子需結束於C或G。 4. 5’端GC含量與3’端GC含量 前6個核苷酸殘基（5,端GC含量）與最後6個核苷酸殘基（3, 端GC含量）具有高GC含量可增進引子專一性。 1293090 a於屬實施例中，例如，定義5，端gc含量观， 且疋義3’端GC含量介於3〇%至6〇%之間。 〜 引子之基本料’獅針對騎標料列的大量 作為候選引子。基於引子本身之序列，本發明之某些實 施例更提供？丨子二級結構之計算。所形成二 算 至少兩個考量因素： 稱之冲异晕" 1·引子之二聚化 CR反應中可能出現兩種引子二聚體，包括自體二聚體與 ，引子二聚體。採用最近相鄰模式（N讓st_NeighbQf爪。㈣（第 1A與1D @)以計算每—種配對情形之穩定性，經由—引子對應 下一引子、順向或反向，依序滑動之方式以審視所有可能的配 對情形’直到重疊長度降到4。對所給引子對之所有配對情形之 評分總數定義為「二聚體評分」。 2.髮夾環（Hairpin loop) 形成髮夾環之強度係以類似方法評估，即，經由使所給引 子之3’端向後「·彎#(bending)」4個核苷酸長並依序滑動其本 身（第1B圖）。髮夾結構之穩定性亦係採用最近相鄰模式，於每 引子之所有暫時滑動配對進行計算。引子對之評分總數定義為 其「髮夾評分」。 本發明之某些實施例更提供引子_至_模板煉合之計算以助 於避免錯誤引導之增幅反應。為避免錯誤引導之增幅反應，引 子之5’端對於標的模板之煉合應比對於3,端之煉合更為穩定。 (第1C圖）。這裡亦可以採用最近相鄰模式評估引子·至_模板之 配對穩定性。在3,端（最後6個鹼基）與5,端（最前6個鹼基）間的 自由能（AG )差異係以下式計算：(3，_5，）= AG 3，- 1293090 例如，將PDA實施例應用在偵測人類中之SARS-CoV時， 候選引子可以設計為避免與人類DNA序列雜交而不去辨認 SARS-CoV之標的基因（第5圖）。 於某些實施型態中，電腦或網路為基的引子設計裝置也提 供一輸出結果。於PDA實施例中，例如，當提出一查詢時，PDA 會回覆一組概要參數（第3A圖）。使用者可以隨著超連結「顯示 最佳之五個引子」或「顯示所有引子」（第3B圖）而由網路瀏覽 器讀出結果。於某些PDA實施例中，使用者可以文字格式下載 以標籤分隔開之查詢結果，或於試算應用軟體如MS-Excel中打 開之。每一查詢序列之最佳引子對將被擷取於「儲存最佳」之 選擇，且所有配對引子對皆擷取至「儲存全部」。 實施例 實施例1 :產生表現序列標籤（EST)種株之引子對 本發明之PDA實施例係用於微陣列合作計晝以產生引子 對，而能增幅儲存於96孔盤之表現序列標籤（EST)種株。一般 而言，種株係以引子由載體臂（vector arms)增幅出來。所增幅之 產物有各種長度，依插入物之大小而異。如此而來，雜交效力 之不一致可能對於各點（spot)強度之量造成嚴重問題。然而，於 批次式模式中，選擇這些EST種株之引子對係受限於增幅產物 之長度。顯然地，除了空的孔洞或錯置的種株，種株成功增幅 的百分比可達99%。 實施例2 :採用或不採用PDA進階選擇以產生針對序列 AP002939之引子對 PDA之系統表現經長度為10,000鹼基之輸入序列 (AP002939:1-10000)以及一 FASTS 格式之 5 序列（約 13,000 鹼基 對）檔案（這些資料組可於pda發現）最佳化處理與測試。當進階 18 1293090 選擇設定為「關」時，完成查詢需花費4·12秒作單一序列之查 詢，與20.21秒作一 5序列之查詢。當該選擇設定為「開」，分 別耗費4.05秒與12.63秒作查詢機制。由於進階選擇之嚴格查 為機制’只有符合資格之候選者可經計算。採用帶有進階選擇 之PDA將得到較少的引子對，且又減少計算時間。考慮到系統 之負載量，提出查詢之序列大小限制在1〇 kb，且一次查詢之總 序列數限制在20。 實施例3 :採用PDA產生對於SARS-CoV基因之新引子組 針對SARS-CoV之敏感性PCR偵測，採用pDA評估引子之適用性 與產生引子序列。採用SARS-CoV之四基因作為候選者以設計新引子 組’上述四基因為：棘⑻(亦稱E2基因’ gi|29826277:21477-25244); 基質(Μ)(亦稱 gi|29826277:26383-27048);核殼(N);以及 〇rflab 聚蛋白 (P)(亦稱 gi|29836505)。 設計外與内引子以助於進行「巢式PCR」。於每一個案中， 選擇500個驗基對長之「外增幅區（outer amplicon)」，而「内 增幅區（inner amplicon)」設為300個驗基對。請參見，例如， 第6圖上方。每一基因之專一性增幅區列示如下： S基因之外增幅區（500 bps): gctaccaaccttacagagttgtagtactttcttttgaacttttaaatgcaccggccacggtttgtg gaccaaaattatccactgaccttattaagaaccagtgtgtcaattttaattttaatggactcactg gtactggtgtgttaactccttcttcaaagagatttcaaccatttcaacaatttggccgtgatgttt ctgatttcactgattccgttcgagatcctaaaacatctgaaatattagacatttcaccttgcgctt ttgggggtgtaagtgtaattacacctggaacaaatgcttcatctgaagttgctgttctatatcaa gatgttaactgcactgatgtttctacagcaattcatgcagatcaactcacaccagcttggcgca tatattctactggaaacaatgtattccagactcaagcaggctgtcttataggagctgagcatgt 1293090 cgacacttcttatgagtgcgacattcctattggagctggcatt (序歹》J 識另號：1) S基因之内增幅區（300 bps): tggccgtgatgtttctgatttcactgattccgttcgagatcctaaaacatctgaaatattagacat ttcaccttgcgcttttgggggtgtaagtgtaattacacctggaacaaatgcttcatctgaagttg ctgttctatatcaagatgttaactgcactgatgtttctacagcaattcatgcagatcaactcaca ccagcttggcgcatatattctactggaaacaatgtattccagactcaagcaggctgtcttatag gagctgagcatgtcgacacttcttatgagtgcgacattcc (序歹識另號：2) M基因之外增幅區（500 bps): accgttgaggagcttaaacaactcctggaacaatggaacctagtaataggtttcctattcctag cctggattatgttactacaatttgcctattctaatcggaacaggtttttgtacataataaagcttgt tttcctctggctcttgtggccagtaacacttgcttgttttgtgcttgctgctgtctacagaattaat tgggtgactggcgggattgcgattgcaatggcttgtattgtaggcttgatgtggcttagctactt cgttgcttccttcaggctgtttgctcgtacccgctcaatgtggtcattcaacccagaaacaaac attcttctcaatgtgcctctccgggggacaattgtgaccagaccgctcatggaaagtgaactt gtcattggtgctgtgatcattcgtggtcacttgcgaatggccggacactccctagggcgctgt gacattaaggacctgccaaaagagatcactgtggctacatcacgaac (序歹識另,J 號: 3) M基因之内增幅區（300 bps) aactcctggaacaatggaacctagtaataggtttcctattcctagcctggattatgttactacaa tttgcctattctaatcggaacaggtttttgtacataataaagcttgttttcctctggctcttgtggc cagtaacacttgcttgttttgtgcttgctgctgtctacagaattaattgggtgactggcgggatt 20 1293090 gcgattgcaatggcttgtattgtaggcttgatgtggcttagctacttcgttgcttccttcaggctg tttgctegtacccgctcaatgtggtcattcaacccag (序歹4 識另,J 號：4) N基因之外增幅區（500 bps) cgtcttggttcacagctctcactcagcatggcaaggaggaacttagattccctcgaggccag ggcgttccaatcaacaccaatagtggtccagatgaccaaattggctactaccgaagagctac ccgacgagttcgtggtggtgacggcaaaatgaaagagctcagccccagatggtacttctatt acctaggaactggcccagaagcttcacttccctacggcgctaacaaagaaggcatcgtatgg gttgcaactgagggagccttgaatacacccaaagaccacattggcacccgcaatcctaataa caatgctgccaccgtgctacaacttcctcaaggaacaacattgccaaaaggcttctacgcag agggaagcagaggcggcagtcaagcctcttctcgctcctcatcacgtagtcgcggtaattca agaaattcaactcctggcagcagtaggggaaattctcctgctcgaatggctagcggaggtgg tgaa (序列識別號：5) N基因之内增幅區（300 bps) catggcaaggaggaacttagattccctcgaggccagggcgttccaatcaacaccaatagtg gtccagatgaccaaattggctactaccgaagagctacccgacgagttcgtggtggtgacggc aaaatgaaagagctcagccccagatggtacttctattacctaggaactggcccagaagcttca cttccctacggcgctaacaaagaaggcatcgtatgggttgcaactgagggagccttgaatac acccaaagaccacattggcacccgcaatcctaataacaatgctgccaccgtg (序歹丨】識 別號：6) P基因之外增幅區（500 bps) 21 1293090 actacgtctatattggcgatcctgctcaattaccagccccccgcacattgctgactaaaggca cactagaaccagaatattttaattcagtgtgcagacttatgaaaacaataggtccagacatgtt ccttggaacttgtcgccgttgtcctgctgaaattgttgacactgtgagtgctttagtttatgacaa taagctaaaagcacacaaggataagtcagctcaatgcttcaaaatgttctacaaaggtgttatt acacatgatgtttcatctgcaatcaacagacctcaaataggcgttgtaagagaatttcttacac gcaatcctgcttggagaaaagctgtttttatctcaccttataattcacagaacgctgtagcttca aaaatcttaggattgcctacgcagactgttgattcatcacagggttctgaatatgactatgtcatTm (°C) = 59.9 + (0.41xGC content) - (675 / primer length) In the PDA embodiment, for example, a preset value is provided, wherein the GC content of the forward and reverse primer ranges from 30% to 70 %. Exclude primers with a Tm value less than 50 °C. 2-Sequence Complexity A low complexity primer reduces the differential energy associated with the sequence content. Therefore, repetition of a single nucleotide must be avoided in both the forward and reverse primers. Continuous dinucleotide repeats such as 'ATATAT' must also be avoided. In PDA embodiments, for example, any four or more repeat (or consecutive) nucleotides, such as AAAA, TTTT, CCCC, or GGGG, must be excluded in both the forward and reverse primers. Continuous dinucleotide repeats such as 'ATATAT' must also be avoided. 3_ 3'-end C/G clamp In the double-strand DNA strand, the nucleotide residue 'C' or 'G' forms a strong paired structure. A stable 3' end in the primer-template complex enhances polymerase potency (Buck, G. A., Fox, J. W., Gunthorpe, M., Hager, Κ·M·, Naeve, C. W., Pon, R. T., Adams, Ρ S. and Rush, J. (1999) Design strategies and performance of custom DNA sequencing primers. Biotechniques, 27, 528-536). Thus, the selected forward or reverse primer tends to have a C or G at the 3' end in the PDA embodiment, for example, a preset 3, end C/G clamp to limit the selection of the forward and reverse primers. End with C or G. 4. The GC content at the 5' end and the GC content at the 3' end are 6 nucleotide residues (5, GC content) and the last 6 nucleotide residues (3, GC content) have high GC content. The introduction is specific. 1293090 a In the examples, for example, the definition of 5, the terminal gc content, and the GC content of the 3' end of the deuterus is between 3% and 6%. ~ The basic material of the introduction lion is a large number of candidates for riding the standard column as a candidate primer. Based on the sequence of the primers themselves, some embodiments of the present invention are provided more? Calculation of the secondary structure of the scorpion. The formation of the second calculation of at least two considerations: called the ambience of heterogeneous "1" dimerization of the introduction of the two reactions in the CR reaction may occur two kinds of primer dimers, including autodimers and primer dimers. Use the nearest neighbor mode (N let st_NeighbQf claws. (4) (1A and 1D @) to calculate the stability of each pairing situation, by means of - primer corresponding to the next primer, forward or reverse, in order to slide Review all possible pairing situations' until the overlap length drops to 4. The total number of scores for all pairs of pairs given is defined as "dimer score". 2. Hairpin loop Forms the strength of the hairpin loop It is evaluated in a similar way, that is, by making the 3' end of the given primer "bending" 4 nucleotides long and sequentially sliding itself (Fig. 1B). Stability of the hairpin structure The nearest neighbor mode is also used to calculate all the temporary sliding pairs of each primer. The total number of scores of the primer pair is defined as the "hairpin score". Some embodiments of the present invention further provide the primer_to_template. Calculate to help avoid false-directed amplification reactions. To avoid false-directed amplification reactions, the 5' end of the primer should be more stable for the calibration of the target template than for the 3, end (Figure 1C). Can also use the nearest The adjacent mode evaluates the pairing stability of the primer to the template. The difference in free energy (AG) between the 3rd end (the last 6 bases) and the 5th end (the first 6 bases) is calculated by the following formula: 3,_5,)= AG 3,- 1293090 For example, when the PDA embodiment is applied to detect SARS-CoV in humans, candidate primers can be designed to avoid hybridization with human DNA sequences without identifying the SARS-CoV target gene. (Figure 5). In some implementations, a computer or network based primer design device also provides an output. In a PDA embodiment, for example, when a query is presented, the PDA will reply to a set of profiles. Parameters (Figure 3A). The user can read the results from the web browser with the hyperlink "Show the best five primers" or "Show all the primers" (Figure 3B). In some PDA embodiments In the user, the user can download the result of the query separated by the label, or open it in the trial application software such as MS-Excel. The best primer pair for each query sequence will be captured in the "storage best" Select, and all paired primer pairs are captured to "storage all EXAMPLES Example 1 : Generation of primers for the expression of sequence tag (EST) species The PDA examples of the invention were used in microarray cooperatives to generate primer pairs, which were amplified and stored in 96-well disks. (EST) Seed plants. In general, the strains are amplified by vector arms with primers. The products of the increase are of various lengths, depending on the size of the insert. As a result, the inconsistency of hybridization may be inconsistent. The amount of intensity for each spot poses a serious problem. However, in batch mode, the choice of primer pairs for these EST species is limited by the length of the amplification product. Obviously, except for empty pores or misplaced For plant strains, the percentage of successful growth of seed plants can reach 99%. Example 2: Advanced selection with or without PDA to generate a primer for PDA of sequence AP002939, an input sequence of 10,000 bases in length (AP002939: 1-1000) and a sequence of 5 FASTS format (approximately 13,000) Base pair) files (these data sets can be found in pda) are optimized for processing and testing. When Advanced 18 1293090 is set to “Off”, it takes 4·12 seconds to complete the query for a single sequence of queries, and 20.21 seconds for a 5 sequence query. When the selection is set to "On", it takes 4.05 seconds and 12.63 seconds respectively to make a query mechanism. Due to the rigorous investigation of advanced selections, only qualified candidates can be calculated. Using a PDA with advanced selection will result in fewer primer pairs and reduced computation time. Considering the load of the system, the sequence size of the proposed query is limited to 1 〇 kb, and the total number of sequences in one query is limited to 20. Example 3: Generation of a new primer set for the SARS-CoV gene using PDA Sensitivity PCR detection for SARS-CoV, pDA evaluation of the applicability of primers and generation of primer sequences. The four genes of SARS-CoV were used as candidates to design a new primer group. The above four genes are: spines (8) (also known as E2 gene 'gi|29826277: 21477-25244); matrix (Μ) (also known as gi|29826277:26383 -27048); nucleocapsid (N); and 〇rflab polyprotein (P) (also known as gi|29836505). Design external and internal primers to facilitate "nested PCR." In each case, select 500 tester pairs for the "outer amplicon" and "inner amplicon" for 300 test pairs. See, for example, above Figure 6. Increased specificity of each region of the gene are as follows: increase in area than the S gene (500 bps): gctaccaaccttacagagttgtagtactttcttttgaacttttaaatgcaccggccacggtttgtg gaccaaaattatccactgaccttattaagaaccagtgtgtcaattttaattttaatggactcactg gtactggtgtgttaactccttcttcaaagagatttcaaccatttcaacaatttggccgtgatgttt ctgatttcactgattccgttcgagatcctaaaacatctgaaatattagacatttcaccttgcgctt ttgggggtgtaagtgtaattacacctggaacaaatgcttcatctgaagttgctgttctatatcaa gatgttaactgcactgatgtttctacagcaattcatgcagatcaactcacaccagcttggcgca tatattctactggaaacaatgtattccagactcaagcaggctgtcttataggagctgagcatgt 1293090 cgacacttcttatgagtgcgacattcctattggagctggcatt (bad sequence "J another identification number: 1) increase the S gene region ( 300 bps): tggccgtgatgtttctgatttcactgattccgttcgagatcctaaaacatctgaaatattagacat ttcaccttgcgcttttgggggtgtaagtgtaattacacctggaacaaatgcttcatctgaagttg ctgttctatatcaagatgttaactgcactgatgtttctacagcaattcatgcagatcaactcaca ccagcttggcgcatatattctactggaaacaatgtattccagactcaagcaggctgtcttatag gagctgagcatgtcgacacttcttatgagtgcgacattcc (bad sequence number identifying another: 2) M gene Growth region (500 bps): accgttgaggagcttaaacaactcctggaacaatggaacctagtaataggtttcctattcctag cctggattatgttactacaatttgcctattctaatcggaacaggtttttgtacataataaagcttgt tttcctctggctcttgtggccagtaacacttgcttgttttgtgcttgctgctgtctacagaattaat tgggtgactggcgggattgcgattgcaatggcttgtattgtaggcttgatgtggcttagctactt cgttgcttccttcaggctgtttgctcgtacccgctcaatgtggtcattcaacccagaaacaaac attcttctcaatgtgcctctccgggggacaattgtgaccagaccgctcatggaaagtgaactt gtcattggtgctgtgatcattcgtggtcacttgcgaatggccggacactccctagggcgctgt gacattaaggacctgccaaaagagatcactgtggctacatcacgaac (Sequence bad identifying another, J No: 3) within the M gene growth region (300 bps) aactcctggaacaatggaacctagtaataggtttcctattcctagcctggattatgttactacaa tttgcctattctaatcggaacaggtttttgtacataataaagcttgttttcctctggctcttgtggc cagtaacacttgcttgttttgtgcttgctgctgtctacagaattaattgggtgactggcgggatt 20 1293090 gcgattgcaatggcttgtattgtaggcttgatgtggcttagctacttcgttgcttccttcaggctg tttgctegtacccgctcaatgtggtcattcaacccag (Sequence bad 4 knowledge another, J: 4) Outside the N gene (500 bps) cgtcttggttcacagctctcactcagcatggca aggaggaacttagattccctcgaggccag ggcgttccaatcaacaccaatagtggtccagatgaccaaattggctactaccgaagagctac ccgacgagttcgtggtggtgacggcaaaatgaaagagctcagccccagatggtacttctatt acctaggaactggcccagaagcttcacttccctacggcgctaacaaagaaggcatcgtatgg gttgcaactgagggagccttgaatacacccaaagaccacattggcacccgcaatcctaataa caatgctgccaccgtgctacaacttcctcaaggaacaacattgccaaaaggcttctacgcag agggaagcagaggcggcagtcaagcctcttctcgctcctcatcacgtagtcgcggtaattca agaaattcaactcctggcagcagtaggggaaattctcctgctcgaatggctagcggaggtgg tgaa: internal (SEQ ID NO 5) N gene growth region (300 bps) catggcaaggaggaacttagattccctcgaggccagggcgttccaatcaacaccaatagtg gtccagatgaccaaattggctactaccgaagagctacccgacgagttcgtggtggtgacggc aaaatgaaagagctcagccccagatggtacttctattacctaggaactggcccagaagcttca cttccctacggcgctaacaaagaaggcatcgtatgggttgcaactgagggagccttgaatac acccaaagaccacattggcacccgcaatcctaataacaatgctgccaccgtg (bad Shu] Sequence ID NO: 6) than the growth region of the P gene (500 bps ) 21 1293090 actacgtctatattggcgatcctgctcaattaccagcccccccacattgctgactaaaggca cactagaaccagaatattttaattc agtgtgcagacttatgaaaacaataggtccagacatgtt ccttggaacttgtcgccgttgtcctgctgaaattgttgacactgtgagtgctttagtttatgacaa taagctaaaagcacacaaggataagtcagctcaatgcttcaaaatgttctacaaaggtgttatt acacatgatgtttcatctgcaatcaacagacctcaaataggcgttgtaagagaatttcttacac gcaatcctgcttggagaaaagctgtttttatctcaccttataattcacagaacgctgtagcttca aaaatcttaggattgcctacgcagactgttgattcatcacagggttctgaatatgactatgtcat
attcacacaaactactgaaacagcacactcttgtaatgtcaaccgcttc (序歹識另,J 號：7) P基因之内增幅區（300 bps) actacgtctatattggcgatcctgctcaattaccagccccccgcacattgctgactaaaggca cactagaaccagaatattttaattcagtgtgcagacttatgaaaacaataggtccagacatgtt ccttggaacttgtcgccgttgtcctgctgaaattgttgacactgtgagtgctttagtttatgacaa taagctaaaagcacacaaggataagtcagctcaatgcttcaaaatgttctacaaaggtgttatt acacatgatgtttcatctgcaatcaacagacctcaaataggcg (序列識別號·· 8) 於每一個案中，在PDA輸入介面設定引子長度為24個鹼 基對，且Tm值為52 °C。為更減低經由雜交導引污染之非 SARS-CoV序列的偽陽性發生，例如，人類序列、候選引子會 以BLAST搜尋序列公開資料庫以核對之（第5圖）。當與已知序 列比對時，選擇獨特的引子以減低偽陽性之可能性。 每一候選基因所得之引子組提供於附件，列示如下： S基因之外增幅區的順向引子：序列識別號：9 ; S基因之外增幅區的反向引子：序列識別號：10 ; 22 1293090 s基因之内增幅區的順向引子：序列識別號： 11 ； s基因之内增幅區的反向引子：序列識別號： 12 ； Μ基因之外增幅區的順向引子：序列識別號： 13 ； Μ基因之外增幅區的反向引子：序列識別號： 14 ； Μ基因之内增幅區的順向引子：序列識別號： 15 ； Μ基因之内增幅區的反向引子：序列識別號： 16 ； Ν基因之外增幅區的順向引子：序列識別號： 17 ； Ν基因之外增幅區的反向引子：序列識別號： 18 ； Ν基因之内增幅區的順向引子：序列識別號： 19 ； Ν基因之内增幅區的反向引子··序列識別號： 20 ; Ρ基因之外增幅區的順向引子：序列識別號： 21 ; Ρ基因之外增幅區的反向引子：序列識別號： 22 ； Ρ基因之内增幅區的順向引子··序列識別號： 23 ； Ρ基因之内增幅區的反向引子：序列識別號： 24 ° 實施例4 :採用所產生之引子進行sars-CoV之偵測 所揭露之任何對引子序列皆可用於SARS_c〇v之债測。可 採用單對（「内引子對」或「外引子對」），或兩組同時使用於同 一 PCR反應以偵測SARS_c〇v。的確，單獨採用外引子對幾乎 與巢式反應系統之敏感性相當。然而，巢式PCR可以得到更有 效之結果。 本發明之組合物可用於各種pCR與巢式pCR ，包括RT與 即時PCR，以偵測DNA或RNA序列（第ό圖）。即時PCR偵測 SARS-CoV 的條件已於 jyh_Lyh Juang 與 shih Sheng Jiang 在 2003年6月30日申請之相關暫時申請案中詳述，其内容併入本 案作為參考資料。採用揭露之引子對，不論單獨或以第一次與 第二次之巢式PCR進行的流程步驟如下所述： 23 1293090 採用單一引子對或巢式RT-PCR之第一次的外引子對時之步驟 流程 採用 Superscript II/白金套組，Invitrogen出品（訂購號 10928-042)或其他類似套組，含有10 μΐ之2倍反應混合物，〇·5 μΐ之50mM MgS04，每引子各取1 μΐ，10 μΜ之反應溶液，以 及0·4 μΐ之RT/Taq混合物。加水至18 μΐ。為得到20 μΐ之總反 應液，加2 μΐ之RNA萃取物至疑似SARS-CoV感染之樣品中。 第一次反應，或當採用單一引子對時之循環如下：45°C ’ 30分鐘；95°C，3分鐘；10循環之95°C，10秒，接著6〇°C， 10秒，並且每循環降溫1°C，然後72°c，20秒；40循環之95 °C，10 秒，52°C，10 秒，72°C，20 秒。 採用巢式RT-PCR第二次反應之内引子組的步驟流程 採用白金級Taq，購自Invitrogen (訂購號10966-018)或其 他類似套組，含有5μ1之10倍白金級Taq反應缓衝液；4μ1之 dNTP混合物，每種核苷酸各2_5mM(使最終每種核苷酸之量為 200μΜ)，2·5μ1 之 50mM MgCl2 ;每 1〇μΜ 之引子取 Ιμΐ ; 〇·2μ1 之白金級Taq，最後加水至49 μΐ。為得到50μ1之總量，加1 0 之第一次PCR產物。 第二次，或稱「巢式」反應之循環為如下：95°C，3分鐘； 10循環之95°C，10秒，接著60°C，10秒，且每循環降溫1°C， 以及之後72°C，20秒；25循環之95°C，10秒，52°C，1〇秒， 72〇C，20 秒。 第7-10圖顯示採用本發明引子組合物於5個人類個體偵測 SARS-CoV所得到的結果，係經由RT-或即時PCR。 簡單地說，樣品係由疑似SARS感染之個體收集，並萃取 病毒RNA。樣品量為1/10至1/30μ1之RNA。所萃取之RNA係 24 1293090 用於RT及即時PCR，或其組合。例如，進行20循環之RT-PCR 後再進行46循環之Q-PCR，共費時3至4小時。 為確認增幅區之大小，由五檢體取得之PCR產物再進行 瓊脂凝膠電泳分析，採用正常人類RNA(由未感染個體取得）作 為陰性對照（第7圖）。 所選引子對於標的SARS-CoV基因之專一性係由Tm分析 檢驗。所選引子與其標的序列煉合溫度顯著地高於與對照組雜 交者（第8圖）。 PCR結果（第9及10圖）確認了低複製數的SARS-CoV仍可 經由本發明方法與組合物而偵測。 對於熟於此技術領域人士而言，本發明之其他實施型態將 經由考量本案說明書與實施例之揭露而更為顯明。本案說明書 與實施例僅係作為例示之用，本發明之實際範圍與精神當視後 附之申請專利範圍而定。 25attcacacaaactactgaaacagcacactcttgtaatgtcaaccgcttc (Sequence identification bad another, J NO: 7) within the P gene growth region (300 bps) actacgtctatattggcgatcctgctcaattaccagccccccgcacattgctgactaaaggca cactagaaccagaatattttaattcagtgtgcagacttatgaaaacaataggtccagacatgtt ccttggaacttgtcgccgttgtcctgctgaaattgttgacactgtgagtgctttagtttatgacaa taagctaaaagcacacaaggataagtcagctcaatgcttcaaaatgttctacaaaggtgttatt acacatgatgtttcatctgcaatcaacagacctcaaataggcg (SEQ ID NO ·· 8) in each case, the PDA input interface 24 is set to a length of primers Base pairs with a Tm value of 52 °C. In order to further reduce the occurrence of false positives of non-SARS-CoV sequences contaminated by hybridization, for example, human sequences and candidate primers will be published in a BLAST search sequence to verify the database (Fig. 5). When compared to known sequences, unique primers are chosen to reduce the likelihood of false positives. The primer set obtained for each candidate gene is provided in the annex, as follows: Forward introduction of the amplification region outside the S gene: sequence identification number: 9; reverse primer of the amplification region outside the S gene: sequence identification number: 10; 22 1293090 cis gene in the amplification region of the s gene: sequence identification number: 11; reverse primer in the amplification region of the s gene: sequence identification number: 12; forward primer in the amplification region outside the gene: sequence identification number : 13 ; Reverse primer of the amplification region outside the Μ gene: sequence identification number: 14 ; cis-inducer of the amplification region within the Μ gene: sequence identification number: 15 ; reverse primer of the amplification region within the Μ gene: sequence recognition No.: 16; Forward introduction of the amplification region outside the Ν gene: sequence identification number: 17; reverse primer of the amplification region outside the Ν gene: sequence identification number: 18; cis-inducer of the amplification region within the Ν gene: sequence Identification number: 19; Reverse primer of the amplification region within the Ν gene··SEQ ID NO: 20; Forward introduction of the amplification region outside the Ρ gene: sequence identification number: 21; Reverse primer of the amplification region outside the Ρ gene : Sequence knowledge No.: 22; cis-inducer of the amplification region within the Ρ gene··SEQ ID NO: 23; Reverse primer for the amplification region within the Ρ gene: Sequence ID: 24 ° Example 4: Using the generated primer for sars Any pair of primer sequences revealed by the -CoV detection can be used for the measurement of SARS_c〇v. A single pair ("inner primer pair" or "external primer pair") can be used, or both groups can be used in the same PCR reaction to detect SARS_c〇v. Indeed, the use of an external primer alone is comparable to the sensitivity of a nested reaction system. However, nested PCR can yield more effective results. The compositions of the invention are useful in a variety of pCR and nested pCRs, including RT and real-time PCR, to detect DNA or RNA sequences (Fig. 3). The conditions for real-time PCR detection of SARS-CoV are detailed in the interim application filed by Jyh_Lyh Juang and shih Sheng Jiang on June 30, 2003, the contents of which are incorporated herein by reference. The procedure for the primer pair, either alone or in the first and second nested PCRs, is as follows: 23 1293090 The first primer pair with a single primer pair or nested RT-PCR The procedure of the procedure is to use Superscript II/Platinum set, Invitrogen (Order No. 10928-042) or other similar kits, containing 10 times the reaction mixture of 10 μM, 50 μM MgS04 of 5 μΐ, 1 μΐ per primer. 10 μΜ of the reaction solution, and an RT/Taq mixture of 0.4 μM. Add water to 18 μΐ. To obtain a total of 20 μL of total reaction solution, add 2 μM of RNA extract to samples suspected of SARS-CoV infection. The first reaction, or when using a single primer pair, is as follows: 45 ° C '30 minutes; 95 ° C, 3 minutes; 10 cycles of 95 ° C, 10 seconds, then 6 ° ° C, 10 seconds, and Each cycle was cooled by 1 ° C, then 72 ° C, 20 seconds; 40 cycles of 95 ° C, 10 seconds, 52 ° C, 10 seconds, 72 ° C, 20 seconds. The procedure of the primer set using the second reaction of nested RT-PCR was performed using Platinum-grade Taq, purchased from Invitrogen (Order No. 10966-018) or other similar kit, containing 5 μl of 10 times platinum-grade Taq reaction buffer; 4 μl of dNTP mixture, each nucleotide 2_5 mM (to make the final amount of each nucleotide is 200 μΜ), 2.5 μl of 50 mM MgCl 2 ; each 1 μ μ μ of the primer is taken ΐ μΐ; 〇 · 2 μ1 of platinum grade Taq, Finally add water to 49 μΐ. To obtain a total of 50 μl, add 1 0 of the first PCR product. The second, or "nested" reaction cycle is as follows: 95 ° C, 3 minutes; 10 cycles of 95 ° C, 10 seconds, followed by 60 ° C, 10 seconds, and each cycle is cooled by 1 ° C, and After 72 ° C, 20 seconds; 25 cycles of 95 ° C, 10 seconds, 52 ° C, 1 〇 second, 72 ° C, 20 seconds. Figures 7-10 show the results obtained by detecting SARS-CoV in five human individuals using the primer composition of the present invention, via RT- or real-time PCR. Briefly, samples are collected from individuals suspected of SARS infection and extract viral RNA. The sample amount is 1/10 to 1/30 μl of RNA. The extracted RNA system 24 1293090 is used for RT and real-time PCR, or a combination thereof. For example, a 20-cycle RT-PCR followed by 46 cycles of Q-PCR took a total of 3 to 4 hours. To confirm the size of the amplification zone, the PCR product obtained from the five samples was subjected to agarose gel electrophoresis analysis using normal human RNA (obtained from uninfected individuals) as a negative control (Fig. 7). The specificity of the selected primer for the standard SARS-CoV gene was tested by Tm analysis. The temperature of the selected primer and its target sequence was significantly higher than that of the control group (Fig. 8). The PCR results (Figures 9 and 10) confirmed that the low copy number of SARS-CoV can still be detected by the method and composition of the present invention. Other embodiments of the present invention will be apparent from the disclosure of the specification and the embodiments of the invention. The description and examples are merely illustrative, and the actual scope and spirit of the invention are intended to be in the scope of the claims. 25