TWI627283B - Method and kit for the field diagnosis of caprine arthritis-encephalitis virus (caev) infection - Google Patents

Method and kit for the field diagnosis of caprine arthritis-encephalitis virus (caev) infection Download PDF

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TWI627283B
TWI627283B TW106110801A TW106110801A TWI627283B TW I627283 B TWI627283 B TW I627283B TW 106110801 A TW106110801 A TW 106110801A TW 106110801 A TW106110801 A TW 106110801A TW I627283 B TWI627283 B TW I627283B
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caev
label
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rpa
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TW201837180A (en
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王佩華
凃柏安
賴芳裕
蕭振文
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國立臺灣大學
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Abstract

本發明提供一種用以偵測山羊關節炎腦炎病毒感染之方法,其包含(a)以重組酶聚合酶擴增CAEV的gag段DNA(nt 618-803);(b)將步驟(a)的擴增產物滴加至一側流層析裝置/試驗片,以側向流至該側流層析裝置/試驗片的遠端,其中該側流層析裝置/試驗片依次包含(i)一樣品墊、(ii)一接合墊、(iii)一檢測區以及(iv)一控制區;及(c)偵測在該檢測區是否有任何擴增產物的結合。另外提供一種用於偵側樣本中山羊關節炎腦炎病毒(CAEV)之套組。本發明提供一種具靈敏度和特異性之RPA-LFD在場診斷方法及套組,以簡單、快速且有成本效益的方法偵測CAEV原病毒DNA。 The present invention provides a method for detecting a goat arthritis encephalitis virus infection, which comprises (a) amplifying a gag segment DNA of CAEV by a recombinase polymerase (nt 618-803); (b) step (a) The amplification product is added dropwise to the one-side flow chromatography device/test piece, and flows laterally to the distal end of the lateral flow chromatography device/test piece, wherein the lateral flow chromatography device/test piece sequentially contains (i) a sample pad, (ii) a bonding pad, (iii) a detection zone, and (iv) a control zone; and (c) detecting whether there is any binding of the amplification product in the detection zone. A kit for the detection of goat arthritis encephalitis virus (CAEV) in the side samples is also provided. The present invention provides a sensitivity and specificity RPA-LFD presence diagnostic method and kit for detecting CAEV proviral DNA in a simple, rapid and cost-effective manner.

Description

在場檢測山羊關節炎腦炎病毒感染之方法及套組 Method and kit for detecting goat arthritis encephalitis virus infection in presence

本發明係關於偵側山羊關節炎腦炎病毒(CAEV)感染之方法及套組,尤其是關於使用重組酶聚合酶擴增側流層析驗棒(RPA-LFD),用以在場進行CAEV偵測診斷之方法及套組。 The present invention relates to a method and a kit for detecting sidearthritic arthritis encephalitis virus (CAEV) infection, in particular for using a recombinase polymerase to amplify a lateral flow chromatography rod (RPA-LFD) for conducting CAEV in the field. Methods and kits for detecting and diagnosing.

山羊關節炎腦炎病毒(CAEV)是小型反芻動物慢病毒(small ruminant lentiviruses,SRLVs)的一種,屬於慢病毒(Lentivirus)屬,反轉錄病毒(Retroviridae)科。該病毒可以感染山羊,有時也感染綿羊和其他相關的反芻動物。流行病學證據指出此病毒的主要感染途徑係小羊經由攝入受CAEV感染成羊之初乳和醫源性傳播,或是長時間親密接觸已感染山羊之橫向傳播。所有的品種和年齡對於該病毒皆為易感性,動物一經感染,則在其生命期間皆為永久性的病毒帶原者。儘管大多數的受感染山羊並無症狀,但所有帶原者持續散布該病毒至環境中,導致未感染山羊中有更多感染。 Goat arthritis encephalitis virus (CAEV) is a type of small ruminant lentiviruses (SRLVs) belonging to the genus Lentivirus and the retroviridae family. The virus can infect goats and sometimes infect sheep and other related ruminants. Epidemiological evidence indicates that the main route of infection of this virus is the colostrum and iatrogenic transmission of lambs infected with CAEV, or the long-term intimate contact with the lateral transmission of infected goats. All breeds and ages are susceptible to the virus, and once the animal is infected, it is a permanent virus carrier during its life. Although most infected goats are asymptomatic, all the originals continue to spread the virus to the environment, resulting in more infections in uninfected goats.

沒有有效的藥物用以治療CAEV的感染,也沒有任何疫苗可預防。因此,準確的診斷後立即從群體中剔除CAEV-陽性動物,為用以降低因疾病造成之潛在經濟損失的主要作法。最常見用於診斷CAE的檢測法是由世界動物衛生組織所建議的血清學檢測法,如:瓊脂凝膠免疫擴散(agar gel immunodiffusion,AGID)試驗,其是基於CAEV血清學。然而,當受感染動物出現延遲血清轉化期時,AGID有低估感染發生的傾向。延遲血清轉化期會阻礙早期和精確的血清學診斷,使可能感染熱源顯現。在台灣,常規的實驗室CAEV感染診斷是基於酵素結合免疫吸附分析法(enzyme-linked immunosorbent assay,ELISA)。在此分析法中,以重組衣殼(capsid,CA)蛋白或包膜醣蛋白次單元的跨膜(transmembrane,TM)域作為抗原,且該分析法已證明比AGID法更為敏感。然而,當受感染動物處於延遲血清轉化期時ELISA同樣有低估感染發生的傾向,且若被試驗動物其被感染的CAEV病毒株與ELISA分析所用的不同,該抗原CA蛋白和TM域的異質性可能導致敏感度不彰。再者,抗原的製備昂貴且耗時,使該分析法用於在場診斷並不實際。 There are no effective drugs to treat CAEV infections, and no vaccines are preventable. Therefore, the removal of CAEV-positive animals from the population immediately after an accurate diagnosis is the main practice to reduce the potential economic loss caused by the disease. The most common test for the diagnosis of CAE is the serological test recommended by the World Organisation for Animal Health, such as: agar gel immunodiffusion (agar) The gel immunodiffusion, AGID) trial, which is based on CAEV serology. However, AGID has a tendency to underestimate infection when infected animals develop a delayed seroconversion phase. Delaying the seroconversion phase can impede early and accurate serological diagnosis, allowing possible infection of the heat source. In Taiwan, conventional laboratory CAEV infection diagnosis is based on enzyme-linked immunosorbent assay (ELISA). In this assay, the transcapsid (TM) domain of the recombinant capsid (CA) protein or envelope glycoprotein subunit is used as the antigen, and this assay has proven to be more sensitive than the AGID method. However, ELISA also has a tendency to underestimate infection when the infected animal is in a delayed seroconversion phase, and if the infected animal has an infected CAEV strain different from that used in the ELISA assay, the heterogeneity of the antigen CA protein and TM domain May cause less sensitivity. Furthermore, antigen preparation is expensive and time consuming, making it impractical for the assay to be used for on-site diagnostics.

因此,仍然需要一個具有高靈敏度及特異性,更低成本、更容易實施、更為全面且快速的方法。 Therefore, there is still a need for a method that is highly sensitive and specific, lower cost, easier to implement, more comprehensive, and faster.

本發明一方面在於提供一種方法,用以偵測山羊關節炎腦炎病毒感染,其包含步驟:(a)於一對正向和反向引子以及一探針序列的存在下,透過重組酶聚合酶擴增(recombinase polymerase amplification,RPA)擴增樣本中CAEV的gag段DNA(nt 512-1858)以作為之標靶序列,該探針序列係互補於該標靶序列的一內部區域,其中一引子具有一第一標記,該探針序列具有一第二標記,使此該標靶序列擴增產生一標記有第一和第二標記之擴增子;(b)將步驟(a)的擴增產物滴加至一側流層析裝置/試驗片(lateral flow device/strip),以側向流至該側流層析裝置/試驗片的遠端,其中該側流層析裝置/試驗片依序包含(i)一樣品墊、(ii)一接合墊、(iii)一檢測區以及(iv)一控 制區,所述接合墊具有以第一作用物標記之移動報告子,該第一作用物可特異性地結合至該擴增子的該第一標記或該第二標記,所述檢測區具有一第二作用物,其特異性地結合該擴增子的該第二標記或該第一標記,以抑制與移動報告子結合的該擴增子進一步地側流層析,所述控制區提供一控制作用物;及(c)偵測在該檢測區是否有任何擴增產物的結合。 One aspect of the invention provides a method for detecting a goat arthritis encephalitis virus infection comprising the steps of: (a) polymerizing through a recombinase in the presence of a pair of forward and reverse primers and a probe sequence Recombinase polymerase amplification (RPA) amplifies the gag segment DNA of CAEV (nt 512-1858) in the sample as a target sequence, and the probe sequence is complementary to an internal region of the target sequence, one of which The primer has a first label, the probe sequence has a second label, such that the target sequence is amplified to produce an amplicon labeled with the first and second labels; (b) the step (a) is expanded The product is added dropwise to a lateral flow device/strip and flows laterally to the distal end of the lateral flow chromatography device/test piece, wherein the lateral flow chromatography device/test piece Included in sequence are (i) a sample pad, (ii) a bonding pad, (iii) a detection zone, and (iv) a control zone, the bonding pad having a movement reporter marked with a first substrate, the first The substrate may specifically bind to the first marker or the second marker of the amplicon, the assay The region has a second substrate that specifically binds to the second label or the first label of the amplicon to inhibit further lateral flow chromatography of the amplicon associated with the mobile reporter, the control The zone provides a control substrate; and (c) detects whether there is any binding of the amplification product in the detection zone.

另一方面是提供一套組,用以偵測樣本中山羊關節炎腦炎病毒,其包含:一對正向和反向引子,以CAEV gag段(nt 512-1858)DNA作為標靶序列為目標;一探針序列,其互補於該標靶序列的一內部區域;一反應劑,用以進行重組酶聚合酶擴增反應;以及一側流層析裝置/試驗片;其中一引子具有一第一標記,該探針序列具有一第二標記,使該標靶序列擴增產生一標記有第一和第二標記之擴增子;且該側流層析裝置/試驗片依序包含(i)一樣品墊、(ii)一接合墊、(iii)一檢測區以及(iv)一控制區,所述接合墊具有以第一作用物標記之移動報告子,該第一作用物其特異性地結合該擴增子的該第一標記或該第二標記,所述檢測區具有一第二作用物,其特異性地結合該第二標記或該第一標記,所述控制區提供一控制作用物。 Another aspect is to provide a set of samples for detecting goat arthritis encephalitis virus in a sample comprising: a pair of forward and reverse primers, with the CAEV gag segment (nt 512-1858) DNA as the target sequence a target; a probe sequence complementary to an internal region of the target sequence; a reagent for performing a recombinase polymerase amplification reaction; and a side stream chromatography device/test piece; wherein the primer has a a first marker, the probe sequence having a second marker, such that the target sequence is amplified to produce an amplicon labeled with the first and second markers; and the lateral flow chromatography device/test strip is sequentially included ( i) a sample pad, (ii) a bonding pad, (iii) a detection zone, and (iv) a control zone, the bonding pad having a movement reporter labeled with a first substrate, the first substrate being specific Sexually binding the first label or the second label of the amplicon, the detection area having a second substrate that specifically binds the second label or the first label, the control region providing a Control the substrate.

結合附圖用以描述非限制性和非詳盡性實施例。應當理解圖式僅描繪了根據本說明書揭示之數個實施例,因此,並非用以限制其範圍,本說明書之揭示將藉由附圖具體詳細描述,其中:圖1為使用102拷貝數的CAEV gag基因重組質體DNA的優選RPA反應溫度。M道和N道分別是分子標記和陰性控制(無DNA水解酶(DNase-free)的水)。 The non-limiting and non-exhaustive embodiments are described in conjunction with the drawings. It should be understood that the drawings depict only disclosed in the specification of several embodiments, therefore, not intended to limit the scope thereof, disclosed in this specification will be described in detail by the accompanying drawings, wherein: Figure 1 is the use of the copy number 102 Preferred RPA reaction temperature for recombinant plastid DNA of CAEV gag gene. M and N are molecular markers and negative controls (DNase-free water, respectively).

圖2為使用天然感染山羊痘病毒(goat pox virus,GPV)和牛白血病病毒(bovine leukaemia virus,BLV)動物的全DNA作為模板進行RPA-AGE(瓊脂糖凝膠電泳法agarose gel electrophoresis,AGE)和RPA-LFD的分子特異性。(A)藉由AGE顯影的結果。(B)藉由LFD顯影的結果。N1:CAEV-陰性山羊萃取的總DNA。N2:陰性控制(無DNA水解酶的水)。 Figure 2 shows RPA-AGE (agarose gel electrophoresis, AGE) using the whole DNA of goats infected with goat pox virus (GPV) and bovine leukaemia virus (BLV) as a template. Molecular specificity of RPA-LFD. (A) Results by AGE development. (B) Results of development by LFD. N1: Total DNA extracted by CAEV-negative goats. N2: Negative control (water without DNA hydrolase).

圖3為使用從CAEV感染山羊中萃取之總DNA以5倍序列稀釋作為模板進行RPA其分子靈敏度測試結果。(A)藉由AGE顯影的結果。(B)相同RPA擴增子藉由LFD顯影的結果。50ng、10ng、2ng、400pg和80pg:從CAEV感染山羊製備之總DNA的連續稀釋;N:無模板控制(無DNA水解酶的水)。 Figure 3 is a graph showing the results of molecular sensitivity test of RPA using 5-fold serial dilution as a template using total DNA extracted from CAEV-infected goats. (A) Results by AGE development. (B) Results of development of the same RPA amplicons by LFD. 50 ng, 10 ng, 2 ng, 400 pg, and 80 pg: serial dilution of total DNA prepared from CAEV-infected goats; N: no template control (water without DNA hydrolase).

圖4為使用10倍序列稀釋質體DNA作為模板其RPA之分子靈敏度測試。(A)藉由AGE顯影的結果,M道和N道:分別為分子標記和陰性控制(無DNA水解酶的水)。(B)相同RPA產物藉由LFD顯影的結果。 Figure 4 is a molecular sensitivity test of RPA using 10-fold serial dilution of plastid DNA as a template. (A) Results of AGE development, M-channel and N-channel: molecular marker and negative control (water without DNA hydrolase). (B) Results of development of the same RPA product by LFD.

以下將參考附圖詳細描述本發明揭示之說明性實施例和示例,藉以使本領域技藝人士可以輕易地實施本發明之構思。 The illustrative embodiments and examples of the present invention are described in detail below with reference to the accompanying drawings, in which FIG.

本文中所稱之「包含或包括」意指不排除一或多個其他組件、步驟、操作和/或元素的存在或添加至所述之組件、步驟、操作和/或元素。「約或接近」或「基本上」意指具有接近於允許指定誤差的數值或範圍,以避免被任何不合理之第三方,違法或不公平的使用為理解本發明揭示之精確或絕對數值。「一」意指該物的語法對象之一個或一個以上(即,至少為一)。 The word "comprising" or "comprises" or "an" or "an" "About or close" or "substantially" means having a value or range that is close to the allowable specified error to avoid any unreasonable use by third parties, illegal or unfair use, to understand the precise or absolute value disclosed herein. "One" means one or more (ie, at least one) of the grammatical objects of the object.

本文揭示之方法可在不使用光學螢光技術下偵測DNA序列(如:病源DNA)。本方法使用RPA分析法和側流層析偵測法分別用以擴增和偵測多種樣品類型中特定DNA序列。該RPA分析法係等溫並且使用噬菌體-提取之重組酶(phage-derived recombinase)UvsX,由其輔因子UvsY協助,該UvsX附聚兩個寡核苷酸引子,以掃描DNA模板中的同源標靶序列。在辨識特定同源序列的情況下,以類似PCR方法,經由金黃色葡萄球菌-提取之DNA聚合酶(Sau DNA polymerase)發生股入侵和其後之股取代擴增,以產生雙股DNA(double-stranded DNA,dsDNA)。 The methods disclosed herein can detect DNA sequences (eg, pathogenic DNA) without the use of optical fluorescence techniques. The method uses RPA analysis and lateral flow chromatography to amplify and detect specific DNA sequences in a variety of sample types, respectively. The RPA assay using phage-based isothermal and - extraction of the recombinant enzyme (phage-derived recombinase) Uvs X , to assist its cofactors Uvs Y, the Uvs X agglomerated two oligonucleotides primers, template DNA to scan Homologous target sequences. In the case of recognizing a specific homologous sequence, a strand-like DNA (double) is generated by a similar PCR method via a S. aureus-extracted DNA polymerase ( Sau DNA polymerase) and subsequent strand-extension amplification. -stranded DNA, dsDNA).

[發明方法][Invention method]

本發明第一方面之方法用以偵測山羊關節炎腦炎病毒感染,其步驟包含:(a)於一對正向和反向引子以及一探針序列的存在下,透過重組酶聚合酶擴增樣本中CAEV的gag段DNA(nt 512-1858)以作為標靶序列,該探針序列係互補於該標靶序列的一內部區域,其中一引子具有一第一標記,該探針序列具有一第二標記,使該標靶序列擴增產生一具有第一和第二標記之擴增子;(b)將步驟(a)的擴增產物滴加至一側流層析裝置/試驗片(lateral flow device/strip),以側向流至該側流層析裝置/試驗片的遠端,其中該側流層析裝置/試驗片依序包含(i)一樣品墊、(ii)一接合墊、(iii)一檢測區以及(iv)一控制區,所述接合墊具有以第一作用物標記之移動報告子,該第一作用物可特異性地結合該擴增子的該第一標記或該第二標記,所述檢測區提供一第二作用物,其特異性地結合該擴增子的該第二標記或該第一標記,以抑制與移動報告子結合的該擴增子進一步地側流層析,所述控制區提供一控制作用物;及(c)偵測在該檢測區是否有任何擴增產物的結合。 The method of the first aspect of the invention is for detecting a goat arthritis encephalitis virus infection, the steps comprising: (a) expanding through a recombinant enzyme polymerase in the presence of a pair of forward and reverse primers and a probe sequence Increasing the gag segment DNA of CAEV (nt 512-1858) in the sample as a target sequence, the probe sequence is complementary to an internal region of the target sequence, wherein one primer has a first marker, and the probe sequence has a second label, the target sequence is amplified to produce an amplicon having first and second labels; (b) the amplification product of step (a) is added dropwise to the one side stream chromatography device/test piece (lateral flow device/strip), flowing laterally to the distal end of the lateral flow chromatography device/test piece, wherein the lateral flow chromatography device/test piece sequentially comprises (i) a sample pad, (ii) a a bonding pad, (iii) a detection zone, and (iv) a control zone, the bonding pad having a movement reporter labeled with a first substrate, the first substrate specifically binding the first of the amplicon a label or the second label, the detection zone providing a second substrate that specifically binds to the first of the amplicon Marking the first marker to inhibit further amplification of the amplicons associated with the mobile reporter, the control region providing a control substrate; and (c) detecting whether there is any expansion in the detection region Combination of product enhancement.

根據本發明,所稱之「樣本」可為一種生物樣本,例如來自於任何疑似帶原CAEV個體的樣本。該樣本可以為感染個體中含有病毒的任何樣本,包含組織和液體樣本,例如:血液、血清、血漿、周邊血液細胞,包含淋巴球和單核細胞、痰、黏膜、尿液、糞便、咽喉拭子樣本、真皮病灶拭子樣本、腦脊髓液、膿以及組織,包含脾、腎和肝。 In accordance with the present invention, a "sample" can be a biological sample, such as from any sample suspected of having a native CAEV. The sample may be any sample containing the virus in an infected individual, including tissue and fluid samples, such as blood, serum, plasma, peripheral blood cells, including lymphocytes and monocytes, sputum, mucous membranes, urine, feces, throat swabs. Subsamples, dermal lesion swab samples, cerebrospinal fluid, pus and tissue, including spleen, kidney and liver.

根據本發明,萃取該樣本的核酸用以進一步評估,較佳地,首先萃取來自該樣本的gDNA,然後通過RPA反應對CAEV之DNA gag段(nt 618-803)進行擴增,其是病毒性基因組的相對保守區。透過設計對gag段(nt 618-803)特異的引子序列,且避免病毒序列中的突變熱點,該較佳的正向引子係具有一SEQ ID NO.1(TCAGAGGGGAGCACTTGACAGAAGGAAATTGT)序列和該反向引子係具有一SEQ ID NO.2(CTAATGTGGCCTGCAAAGACATAAAGTCT)序列。此外,其中一引子具有一第一標記。當在擴增反應期間擴增該DNA標靶序列時,一具有第二標記之探針特異性地與擴增子內的內部序列雜交,如此該擴增子會帶有第一標記和第二標記,其中該第二標記不同於第一標記。更佳地,該探針序列係由具有5’端FAM(5’-FAM)抗原標記的SEQ ID NO.3(GGGGAGCACTTGACAGAAGGAAATTGTTTA),且以THF間隔子(THF spacer)連接至一在3’終端帶有一C3間隔子(C3-spacer)的SEQ ID NO.4(GGTGCCTTAAAACAT)序列下游。 According to the present invention, the nucleic acid of the sample is extracted for further evaluation. Preferably, the gDNA from the sample is first extracted, and then the DNA gag segment of CAEV (nt 618-803) is amplified by an RPA reaction, which is viral. A relatively conserved region of the genome. By designing a primer sequence specific for the gag segment (nt 618-803) and avoiding mutation hotspots in the viral sequence, the preferred forward primer has a sequence of SEQ ID NO. 1 (TCAGAGGGGAGCACTTGACAGAAGGAAATTGT) and the reverse primer system. There is a sequence of SEQ ID NO. 2 (CTAATGTGGCCTGCAAAGACATAAAGTCT). In addition, one of the primers has a first mark. When the DNA target sequence is amplified during the amplification reaction, a probe having a second label specifically hybridizes to an internal sequence within the amplicon, such that the amplicon carries a first label and a second a mark, wherein the second mark is different from the first mark. More preferably, the probe sequence is SEQ ID NO. 3 (GGGGAGCACTTGACAGAAGGAAATTGTTTA) having a 5'-end FAM (5'-FAM) antigen label and is ligated to a 3' terminal strip with a THF spacer (THF spacer). There is a C3 spacer (C3-spacer) downstream of the SEQ ID NO. 4 (GGTGCCTTAAAACAT) sequence.

較佳地,該第一和第二標記係選自於半抗原(haptens)例如:生物素(biotin)、螢光素衍生物(如:FITC或FAM)、玫瑰紅衍生物(如:TAMRA)、級聯藍(Cascade Blue)、螢光黃(Lucifer yellow)、5-溴-2-去氧尿苷 (5-bromo-2-deoxyuridine,BrdU)、二硝基酚(dinitrophenol,DNP)、地谷新配質(digoxygenin,DIG),及短肽標記序列(即,可以產生特異性抗體的短肽)。更佳地,該第一標記係生物素,該第二標記係FAM,在這種情況下,在該擴增步驟中產生的擴增子會標記有生物素和FAM。 Preferably, the first and second markers are selected from haptens such as biotin, luciferin derivatives (eg FITC or FAM), rose red derivatives (eg TAMRA) , Cascade Blue, Lucifer yellow, 5-bromo-2-deoxyuridine (5-bromo-2-deoxyuridine, BrdU), dinitrophenol (DNP), digoxygenin (DIG), and short peptide tag sequences (ie, short peptides that produce specific antibodies) . More preferably, the first marker is biotin, the second marker is FAM, in which case the amplicon produced in the amplification step will be labeled with biotin and FAM.

接續於擴增步驟之後,該擴增產物滴加至一側流層析裝置/試驗片。該側流層析裝置/試驗片包含一基質,其可讓加入緩衝液之該擴增產物持續的毛細作用或側向層析流至該側流層析裝置的遠端。一般而言,該側流層析裝置之該基質為試驗片的形式。較佳地,該基質係由硝化纖維素膜、聚二氟亞乙烯(polyvinylidene fluoride,PVDF)、尼龍或單一多孔材料基材所組成。該側流層析裝置在近端包含一樣本墊和一接合墊,在遠端或是鄰近遠端包含一檢測區(也稱為檢測線)和一控制區(也稱為控制線)。該樣本墊係用以承載擴增產物,且當該擴增產物側向流至該接合墊,在接合墊中之具有一第一作用物的移動報告子將會特異性地結合至該擴增子的該第一標記或該第二標記。 Following the amplification step, the amplification product was added dropwise to the one-side flow chromatography device/test piece. The lateral flow chromatography device/test strip comprises a matrix that allows continuous capillary or lateral chromatography of the amplification product added to the buffer to the distal end of the lateral flow chromatography device. In general, the matrix of the lateral flow chromatography device is in the form of a test strip. Preferably, the matrix consists of a nitrocellulose membrane, polyvinylidene fluoride (PVDF), nylon or a single porous material substrate. The lateral flow chromatography device includes a native pad and a bonding pad at the proximal end and a detection zone (also referred to as a detection line) and a control zone (also referred to as a control line) at the distal end or adjacent distal end. The sample pad is configured to carry an amplification product, and when the amplification product flows laterally to the bonding pad, a mobile reporter having a first substrate in the bonding pad will specifically bind to the amplification The first mark or the second mark of the child.

所稱之「移動報告子」係指微粒,其可由廣泛的各種物質組成,但較佳係由一種或多種基本上惰性的物質所組成,如:金、二氧化矽(silica)、硒、聚苯乙烯(polystyrene)、三聚氰胺樹脂(melamine resin)、聚甲基丙烯酸鹽(polymethacrylate)、苯乙烯/二乙烯苯(styrene/divinylbenzene)共聚物或是聚乙烯基甲苯(polyvinyltoluene)等標記有第一作用物之微粒。該微粒較佳係非多孔的。該微粒可包含一物質讓側流層析裝置上檢測區和控制區的結果顯像。更便利地,此一物質將係一種染料或其他有色物質,以使肉眼可目視效果,然而,或者該物質例如可為一種標記物質使其可通過產生 一有色物質(如:酵素或其他催化標記物)或藉由螢光、發光或磁力作用(如:使用螢光計、光度計或磁感應)顯像。 By "mobile reporter" is meant microparticles which may be composed of a wide variety of materials, but are preferably composed of one or more substantially inert materials such as gold, silica, selenium, poly Polystyrene, melamine resin, polymethacrylate, styrene/divinylbenzene copolymer or polyvinyltoluene have the first effect Particles of matter. The particles are preferably non-porous. The microparticles may comprise a substance for imaging the results of the detection zone and the control zone on the lateral flow chromatography device. More conveniently, the substance will be a dye or other colored substance to give the naked eye a visual effect, however, or the substance may be, for example, a labeling substance such that it can be produced. A colored substance (eg, an enzyme or other catalytic label) is imaged by fluorescence, luminescence, or magnetic force (eg, using a fluorometer, luminometer, or magnetic induction).

該第一作用物係選自具有該第一標記或該第二標記且有特異性結合或反應能力的作用物。因此,該第一作用物可以是一種抗體、抗體片段(如:Fab、F(ab')2和scfv片段)、受體或其他結合配偶體。該第一作用物本身可與酵素或催化物質接合,使偵測產物顯像而受偵測。當該第一標記係生物素且該第一作用物設計用以該第一標記結合,則該第一作用物可為鏈黴親合素(streptavidin)或抗生物素蛋白(avidin),但更佳係一種抗生物素(anti-biotin)抗體。 The first substrate is selected from the group consisting of the first label or the second label and having the ability to specifically bind or react. Thus, the first agent can be an antibody, an antibody fragment (eg, a Fab, F(ab ' )2, and a scfv fragment), a receptor, or other binding partner. The first substrate itself can be bound to the enzyme or catalytic material to cause the detected product to be visualized and detected. When the first label is biotin and the first substrate is designed to bind to the first label, the first substrate may be streptavidin or avidin, but more An anti-biotin antibody.

該檢測區係提供一第二作用物,其可特異性地結合所述第二標記或所述第一標記。第二作用物結合該第二標記或該第一標記是取決於該第一作用物且應不同於該第一作用物的該結合配體。例如:若該第一作用物係設計用以結合該第一標記,則該第二作用物應結合該第二標記。較佳地,該第一標記係生物素,該第二標記係FAM,且該第一作用物係設計用以結合該第二標記,則該第一作用物可以是抗FAM(anti-FAM)抗體,且該第二作用物可以是鏈黴親和素、抗生物素蛋白或一種抗生物素抗體。 The detection zone provides a second substrate that specifically binds the second marker or the first marker. The second agent binds to the second label or the first label is dependent on the first substrate and should be different from the binding partner of the first substrate. For example, if the first substrate is designed to bind the first marker, the second substrate should be combined with the second marker. Preferably, the first label is biotin, the second label is FAM, and the first substrate is designed to bind the second label, and the first object may be anti-FAM (anti-FAM) An antibody, and the second agent can be streptavidin, avidin or an anti-biotin antibody.

該檢測區提供檢測結果。即,該檢測區利用第二作用物,結合第一或第二標記從而固定擴增子(其中每一擴增子應結合至移動報告子),因此可顯示樣本中是否具有預偵測的CAEV原病毒之結果。「陽性」(即,檢測結果指出在樣本中有CAEV原病毒)檢測結果較佳係由在檢測區中顯現可目視的顏色訊號,該顏色訊號係由該移動報告子提供。當該移動報告子為金微粒,該可目視顏色訊號將會是略帶粉色的紅色。 The detection zone provides detection results. That is, the detection zone uses a second substrate, in combination with the first or second label to immobilize the amplicon (where each amplicon should be bound to the mobile reporter), thereby displaying whether the sample has a pre-detected CAEV The result of the original virus. The "positive" (i.e., the result of the test indicates that there is a CAEV provirus in the sample) is preferably caused by a visual color signal appearing in the detection zone, the color signal being provided by the mobile reporter. When the mobile report is gold particles, the visual color signal will be a slightly pinkish red.

該控制區係側流層析裝置/試驗片上有別於檢測區的一個區域。較佳地,該控制區係位於該檢測區和該側流層析裝置/試驗片的該遠端之間。該控制區提供一陽性控制結果。如後所述,該陽性控制結果可顯示該移動報告子已成功流過該試驗片。因此,在最簡單的情況下,該控制區係提供一控制作用物,其特異性地結合該第一作用物,在這種情況下,該控制區結合且固定該移動報告子,從而提供一結果顯示該移動報告子是否成功流過試驗片上的基質。 The control zone is a region of the lateral flow chromatography device/test piece that is different from the detection zone. Preferably, the control zone is located between the detection zone and the distal end of the lateral flow chromatography device/test strip. The control zone provides a positive control result. As will be described later, the positive control result may indicate that the mobile reporter has successfully flowed through the test piece. Thus, in the simplest case, the control zone provides a control substrate that specifically binds the first substrate, in which case the control zone combines and immobilizes the mobile reporter to provide a The results show whether the mobile reporter successfully passed through the substrate on the test piece.

[本發明之套組][Set of the invention]

本發明也包含一套組用以進行根據本發明所述之該方法。 The invention also encompasses a set of methods for carrying out the method according to the invention.

本文所稱之「套組」係關於一套反應劑的組合,適用於根據本發明之方法偵測一標靶DNA,結合一或多個類型的元件或組成(例如:其他類型的生化反應劑、容器、適合用以商業販售之包裝、該反應劑限制的基質、電子硬體組件等)。 As used herein, a "set" is a combination of reagents suitable for detecting a target DNA in accordance with the methods of the invention, in combination with one or more types of elements or compositions (eg, other types of biochemical reactants) , containers, packages suitable for commercial sale, substrates for such reactants, electronic hardware components, etc.).

本發明第二方面是關於該套組,其用以偵測樣本中山羊關節炎腦炎病毒(CAEV),其包含:一對正向和反向引子,以CAEV gag段(nt 512-1858)DNA作為標靶序列為目標;一探針序列,其互補於該標靶序列的一內部區域;一反應劑,用以進行重組酶聚合酶擴增反應;以及一側流層析裝置/試驗片;其中一引子具有一第一標記,該探針序列具有一第二標記,使該標靶序列擴增產生一標記有第一和第二標記之擴增子;且該側流層析裝置/試驗片依序包含(i)一樣品墊、(ii)一接合墊、(iii)一檢測區以及(iv)一控制區,所述接合墊具有以第一作用物標記之移動報告子,該第一作用物可特異性地結合該擴增子的該第一標記或該第二標記,所述檢測區提供一第 二作用物,其特異性結合該第二標記或該第一標記,所述控制區提供一控制作用物。 A second aspect of the invention relates to the kit for detecting a goat arthritis encephalitis virus (CAEV) in a sample comprising: a pair of forward and reverse primers, with a CAEV gag segment (nt 512-1858) DNA as a target sequence; a probe sequence complementary to an internal region of the target sequence; a reagent for performing a recombinase polymerase amplification reaction; and a side stream chromatography device/test piece One of the primers has a first label, the probe sequence has a second label, and the target sequence is amplified to generate an amplicon labeled with the first and second labels; and the lateral flow chromatography device/ The test piece sequentially comprises (i) a sample pad, (ii) a bonding pad, (iii) a detection zone, and (iv) a control zone, the bonding pad having a movement reporter marked with a first substrate, the test piece The first substrate can specifically bind to the first label or the second label of the amplicon, and the detection region provides a first A second substrate that specifically binds to the second label or the first label, the control region providing a control substrate.

本文所述之「重組酶聚合酶擴增」係一種藉由重組酶作用物至目標寡核苷酸以標靶DNA,結合由聚合酶以合成DNA之方法。首先,一重組酶作用物與一第一和第二核酸引子接觸,以形成一第一和第二核酸蛋白引子。接著,該第一和第二核酸蛋白引子與雙股標靶序列接觸,在所述第一股的第一部分形成一第一雙股結構,且在所述第二股的第二部分形成一第二雙股結構,所以所述第一核酸引子的該3’終端和所述第二核酸引子係目標朝向各個所已知的模板DNA分子。第三,所述第一和第二核酸蛋白引子的該3’終端藉由DNA聚合酶用以產生第一和第二雙股核酸,以及核酸的第一和第二置換股。最後,重複該第二和第三步驟直至達到所需的擴增度。RPA所需的反應劑如美國專利US 2015/0240298A1所述,其整體在此以參考方式包括在內。 As used herein, "recombinase polymerase amplification" is a method of synthesizing DNA by binding a recombinant enzyme substrate to a target oligonucleotide to target DNA. First, a recombinase substrate is contacted with a first and second nucleic acid primer to form a first and second nucleic acid protein primer. Next, the first and second nucleic acid protein primers are contacted with the double-strand target sequence, forming a first double-strand structure in the first portion of the first strand, and forming a first portion in the second portion of the second strand The double-stranded structure, such that the 3' terminal and the second nucleic acid primer of the first nucleic acid primer are directed toward each of the known template DNA molecules. Third, the 3' terminal of the first and second nucleic acid protein primers is used by the DNA polymerase to generate the first and second double stranded nucleic acids, as well as the first and second replacement strands of the nucleic acid. Finally, the second and third steps are repeated until the desired degree of amplification is achieved. Reagents required for RPA are described in U.S. Patent No. US 2015/0240298 A1, the entire disclosure of which is incorporated herein by reference.

[實施例][Examples]

以下本文揭示將以參考實施例與圖式具體描述。然而,本文揭示並非限制於所述實施例及圖式。 The following disclosure will be specifically described with reference to the embodiments and drawings. However, the disclosure herein is not limited to the embodiments and the drawings.

[材料與方法][Materials and Methods]

動物和血液樣本Animal and blood samples

血液樣本是從台灣不同地區不同品種(阿爾拜因山羊94隻、撒能36隻及奴比亞山羊70隻)之3個群體所收集而來,其中一個群體參與臺灣農委會指導之國家山羊關節炎腦炎(CAEV)疾病防治及清除計畫。其他兩個群體同時為之前進行過血清學檢測CAEV陽性之群體。以如下所述之ELISA 試驗,檢測具有或不具有抗-CAEV抗體之血清陰性和血清陽性的山羊進行取樣。包括在血清陽性動物中表現出慢性關節炎(“大膝症”)的許多山羊,此為典型CAEV感染之症狀。藉由頸靜脈穿刺同時收集200隻山羊中每一隻之血清樣本和EDTA-抗凝血樣本。此動物流程由行政院農委會畜牧研究所新竹分所之實驗動物倫理委員會批准。 The blood samples were collected from three groups of different species in different parts of Taiwan (94 Albay goats, 36 Saaneng and 70 slaves), one of which participated in the national goats guided by the Taiwan Council of Agriculture. Arthritis encephalitis (CAEV) disease prevention and elimination program. The other two groups were also those who had previously been serologically tested for CAEV positive. ELISA as described below Tests were performed to detect seronegative and seropositive goats with or without anti-CAEV antibodies for sampling. Includes many goats that exhibit chronic arthritis ("big knees") in seropositive animals, a symptom of a typical CAEV infection. Serum samples and EDTA-anticoagulant samples from each of 200 goats were simultaneously collected by jugular vein puncture. This animal procedure was approved by the Experimental Animal Ethics Committee of the Hsinchu Branch of the Animal Research Institute of the Executive Yuan.

血清學CAEV診斷試驗Serological CAEV diagnostic test

血清是由凝結的血液樣本經1,200 x g轉速離心20分鐘收集而來,並於-20℃保存至使用前。利用市售的ELISA套組分析CAEV抗體是否存在,使用來自Bommeli Diagnostics公司(瑞士里貝菲爾德-伯恩)的Checkit梅迪-威司奈病毒(maedi-visna virus,MVV)/CAEV全病毒ELISA分析。該檢測按照製造商之說明書進行。 Serum was collected from a condensed blood sample by centrifugation at 1,200 x g for 20 minutes and stored at -20 °C until use. Analysis of the presence of CAEV antibodies using a commercially available ELISA kit, using the Checkit medi-visna virus (MVV)/CAEV whole virus ELISA assay from Bommeli Diagnostics (Ribery-Bern, Switzerland) . The test is carried out in accordance with the manufacturer's instructions.

總DNA模板製備Total DNA template preparation

使用QIAamp DNA Mini套組(Qiagen公司,法國科塔布夫)按照製造商的說明書,萃取EDTA-抗凝血中的總DNA。使用Nano Drop 1000分光光度計(Thermo Fisher Scientific Inc.,美國麻州沃爾瑟姆)在260nm和280nm下,測量DNA的濃度和品質,然後用不含DNA水解酶(DNase-free)的水調整至50ng/μL。將該定量的DNA保存於-20℃中直到進行RPA反應。在優選的RPA-LFD反應中使用1微升的模板。從細胞培養中萃取出山羊痘病毒DNA和牛白血病病毒RNA。按照Rovnak和Casey(1999)的方法藉由反轉錄酶PCR以擴增牛白血病病毒RNA。 Total DNA in EDTA-anticoagulation was extracted using a QIAamp DNA Mini kit (Qiagen, Cotabov, France) according to the manufacturer's instructions. The concentration and quality of the DNA were measured at 260 nm and 280 nm using a Nano Drop 1000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA), and then adjusted with water without DNA hydrolase (DNase-free). Up to 50 ng/μL. This quantitative DNA was stored at -20 ° C until the RPA reaction was carried out. One microliter of template was used in the preferred RPA-LFD reaction. Goat poxvirus DNA and bovine leukemia virus RNA were extracted from cell culture. Bovine leukemia virus RNA was amplified by reverse transcriptase PCR according to the method of Rovnak and Casey (1999).

重組質體建構Recombinant plastid construction

訂購和合成位於CAEV原病毒基因組nt512和nt1858位置間 的CAEV gag基因序列(GenBank登錄號:M33677.1),然後用以複製至Integrated DNA Technologies公司(IDT,美國愛荷華州科勒爾維爾)之pUCIDT-AMP載體。根據製造商的質體再懸浮方案,將該重組質體離心並再懸浮於80μL的IDTE(10mM Tris,0.1mM EDTA)緩衝液(pH 7.5-8.0),以達到濃度約50ng/μL作為儲備濃度,並保存於-20℃至使用前。在每個RPA反應中使用兩微升的模板。 Order and synthesis located between the CAEV proviral genomes nt512 and nt1858 The CAEV gag gene sequence (GenBank accession number: M33677.1) was then replicated to the pUCIDT-AMP vector of Integrated DNA Technologies (IDT, Coralville, Iowa, USA). The recombinant plasmid was centrifuged and resuspended in 80 μL of IDTE (10 mM Tris, 0.1 mM EDTA) buffer (pH 7.5-8.0) according to the manufacturer's plastid resuspension protocol to achieve a concentration of approximately 50 ng/μL as a stock concentration. And stored at -20 ° C until use. Two microliters of template was used in each RPA reaction.

重組酶聚合酶擴增引子和探針設計Recombinase polymerase amplification primer and probe design

因為RPA詳細機制尚未完全瞭解,已知的寡核苷酸引子的擴增性能純粹基於其序列,建議將軟體設計之引子經過檢測開發程序,以篩選一系列的候選引子,並選擇出較佳的引子對。根據TwistAmp®反應套組手冊中的附錄,一系列合適(29-35 bp,GC含量30-70%)之候選RPA引子是藉由Primer3以CAEV gag基因(512-1858 nt,GenBank登錄號:M33677.1)DNA序列中的保守區為目標所設計而得。依照TwistAmp反應套組手冊以篩選引子,用於檢測CAEV gag基因之保守區(nt 618-803,186 bp)的該一般正向引子P001(SEQ ID NO.1,5'-TCAGAGGGGAGCACTTGACAGAAGGAAATTGT-3',nt 618-649)和具有5’端生物素標記(5'-biotin)的該反向引子P002(5'-biotin-CTAATGTGGCCTGCAAAGACATAAAGTCT(SEQ ID NO.2)-3',nt 775-803),使用TwistAmp® Basic套組(TwistDx,英國劍橋)能滿足發明所需的要求。之後,用於LFD分析法的兩個nfo DNA探針(LF探針1和LF探針2)是由RPA引子間的序列設計而得。該探針由攜帶5'-FAM抗原標記的上游段(30 nt),並藉由THF間隔子連接到在其3’終端攜帶C3-間隔物(聚合酶擴張阻 斷基團)的相鄰下游寡核苷酸(15 nt)所組成。使用TwistAmp® nfo套組(TwistDx,英國)測試該設計的nfo探針其與該正向P001和該反向P002引子的相容性,以擴增該CAEV gag基因(nt 512-1858)。該LF探針1(5'-FAM-GGGGAGCACTTGACAGAAGGAAATTGTTTA(SEQ ID NO.3)-THF-GGTGCCTTAAAACAT(SEQ ID NO.4)-C3-spacer-3')表現出最佳的標靶擴增(183 bp)。該引子和該探針是由Integrated DNA Technologies公司(IDT,美國愛荷華州科勒爾維爾)合成。 Because the detailed mechanism of RPA is not fully understood, the amplification performance of known oligonucleotide primers is purely based on its sequence. It is recommended that the software design primers be tested and developed to screen a series of candidate primers and select better ones. Lead pair. The reaction TwistAmp ® kit Manual Appendix, the appropriate range (29-35 bp, GC content of 30-70%) of the primer is by RPA candidate Primer3 to CAEV gag gene (512-1858 nt, GenBank accession number: M33677 .1) Conserved regions in the DNA sequence are designed for the target. The general forward primer P001 (SEQ ID NO. 1, 5 ' -TCAGAGGGGAGCACTTGACAGAAGGAAATTGT-3 ' , nt 618 was used to screen the conserved region of the CAEV gag gene (nt 618-803, 186 bp) according to the TwistAmp Reaction Kit Manual. '(this reverse primer P002 -biotin) of (5' -biotin-CTAATGTGGCCTGCAAAGACATAAAGTCT (SEQ ID NO.2 -3 -649) and having a 55) 'end biotin-labeled', nt 775-803), using TwistAmp ® The Basic set (TwistDx, Cambridge, UK) meets the requirements of the invention. Thereafter, two nfo DNA probes (LF probe 1 and LF probe 2) for LFD analysis were designed from the sequence between the RPA primers. The probe is flanked by an upstream stretch (30 nt) carrying the 5 ' -FAM antigen and ligated to the adjacent downstream of the C3-spacer (polymerase expansion blocking group) at its 3' end by a THF spacer. It consists of an oligonucleotide (15 nt). Use TwistAmp ® nfo kit (TwistDx, UK) probes to test the design nfo with the forward and the backward compatibility P001 P002 primers to amplify the CAEV gag gene (nt 512-1858). The LF probe 1 (5 ' -FAM-GGGGAGCACTTGACAGAAGGAAATTGTTTA (SEQ ID NO. 3)-THF-GGTGCCTTAAAACAT (SEQ ID NO. 4)-C3-spacer-3 ' ) showed the best target amplification (183 bp) ). The primer and probe were synthesized by Integrated DNA Technologies (IDT, Coralville, Iowa, USA).

重組酶聚合酶擴增條件和優化Recombinase polymerase amplification conditions and optimization

對於引子的篩選,略微修改使用TwistAmp® Basic套組(TwistDx,英國劍橋)對製造商的方案,以50μL體積進行RPA反應。將1ng重組質體DNA、0.42μM每種RPA引子、14mM醋酸鎂、1×復水緩衝液(rehydration buffer)以及不含DNA水解酶(DNase-free)的水的混合物,加到乾燥酵素顆粒中,並充分混合。將醋酸鎂移液至管蓋,隨後將醋酸鎂離心進入管中,在37℃下引發該RPA機制反應60分鐘。該反應透過4%瓊脂糖凝膠電泳(AGE)顯影。對於探針的篩選,使用TwistAmp® nfo試劑盒(TwistDx,英國劍橋)以50μL體積進行RPA反應。透過以5'端生物素(5' biotin)殘基標記反向P002引子使P001和P002引子適用於RPA分析,並測試這兩種引子與根據TwistDX指南所設計的內部RPA LF探針(LF探針1或LF探針2)的相容性。接著將0.42μM的每種RPA引子(P001和P002)及0.12μM的LF探針(LF探針1或LF探針2)以1×復水緩衝液和不含DNA水解酶的水混合,然後將該混合物加進TwistAmp nfo套組(TwistDx,英國劍橋)的乾燥顆粒。加入該重組質體DNA(1ng),並將14mM的醋酸鎂移液到管蓋,之後在37℃下進行離心以引 發該RPA機制反應60分鐘。該反應藉由4%AGE顯影。 For screening of the primer, using slightly modified TwistAmp ® Basic kit (TwistDx, Cambridge, UK) to the manufacturer's protocol, for RPA reaction volume to 50μL. A mixture of 1 ng of recombinant plastid DNA, 0.42 μM of each RPA primer, 14 mM magnesium acetate, 1×rehydration buffer, and water without DNA hydrolase (DNase-free) was added to the dried enzyme granules. And mix well. The magnesium acetate was pipetted to the tube lid, and then magnesium acetate was centrifuged into the tube, and the RPA mechanism was initiated at 37 ° C for 60 minutes. The reaction was developed by 4% agarose gel electrophoresis (AGE). For screening probe using TwistAmp ® nfo kit (TwistDx, Cambridge, UK) in 50μL reaction volume of RPA. Through to the 5 'end biotin (5' Biotin) residues labeled reverse primers P002 and P002 P001 make RPA primer suitable for analysis, and testing with the two primers (LF RPA probe according to the LF internal probe designed guide TwistDX Compatibility of needle 1 or LF probe 2). Next, 0.42 μM of each RPA primer (P001 and P002) and 0.12 μM of LF probe (LF probe 1 or LF probe 2) were mixed with 1×rehydration buffer and DNA without hydrolase, and then The mixture was added to dry granules of a Twist Amp nfo kit (TwistDx, Cambridge, UK). The recombinant plastid DNA (1 ng) was added, and 14 mM magnesium acetate was pipetted into the cap, followed by centrifugation at 37 ° C to initiate the RPA mechanism reaction for 60 minutes. The reaction was developed by 4% AGE.

對於在場樣本的CAEV診斷,使用TwistAmp® nfo套組(TwistDx,英國劍橋)以50μL體積進行RPA反應。首先混合1μL的現場基因組DNA樣本、0.42μM每種RPA引子(P001+P002引子)、0.12μM的LF探針1、14mM醋酸鎂、1×復水緩衝液以及不含DNA水解酶的水,然後加到乾燥酵素顆粒。將醋酸鎂移液到管蓋,然後關閉該蓋子,隨後將醋酸鎂經離心進入管中以引發該RPA機制。將該試管在37℃下培養30分鐘。 For diagnosis of the presence of CAEV samples, using TwistAmp ® nfo kit (TwistDx, Cambridge, UK) in 50μL reaction volume of RPA. First mix 1 μL of the field genomic DNA sample, 0.42 μM each RPA primer (P001 + P002 primer), 0.12 μM LF probe 1, 14 mM magnesium acetate, 1 × rehydration buffer, and DNA without DNA hydrolase, then Add to dry enzyme granules. The magnesium acetate was pipetted to the cap, then the lid was closed, and then magnesium acetate was centrifuged into the tube to initiate the RPA mechanism. The tube was incubated at 37 ° C for 30 minutes.

側流層析試紙(Lateral flow dipstick,LFD)分析法Lateral flow dipstick (LFD) analysis

該RPA擴增子可在LFD試驗片(Milenia Biotec,德國吉森)上觀察到顯示為陽性檢測線。為在LFD上檢測該RPA擴增物,將2μL的RPA產物加到含有120μL分析緩衝液(具有0.1% Tween 20的1×磷酸鹽緩衝鹽水)的新試管中。然後將LFD試驗片浸入該混合物中5分鐘以顯像檢測結果。即,將該LFD試驗片浸入混合物中之後,混合物的組成可使其流過該膜,其包含一樣本墊、一接合墊、一檢測區及一控制區。該接合墊具有移動報告子,其帶有抗-FAM抗體,因此該移動報告子可與雙重標記FAM和生物素的擴增子結合。該檢測線包含抗-生物素抗體,「捕集」雙重標記的擴增子。因雙重標記擴增子與移動報告子結合,因此,其在檢測線上被抗-生物素抗體捕集的情況下,會產生一略帶粉色的紅色線。另一方面,該陽性控制線包含次級抗-FAM抗體,會捕集無論是未與擴增子結合移動報告子,或是未被檢測線上的抗-生物素抗體捕捉與擴稱子結合的移動報告子。該陽性控制線用以確認該移動報告子已流過該膜。在該陽性控制線捕集移動報告子,也會產生一略帶粉色的紅色線。該整體LFD分析是在室溫下進行。 The RPA amplicon was observed to be a positive line on the LFD test piece (Milenia Biotec, Giessen, Germany). To detect the RPA amplification on LFD, 2 [mu]L of RPA product was added to a new tube containing 120 [mu]L of assay buffer (1 x phosphate buffered saline with 0.1% Tween 20). The LFD test piece was then immersed in the mixture for 5 minutes to visualize the test results. That is, after the LFD test piece is immersed in the mixture, the composition of the mixture can flow through the film, which comprises the same pad, a bonding pad, a detection zone, and a control zone. The mat has a mobile reporter with an anti-FAM antibody so the mobile reporter can bind to the double labeled FAM and biotin amplicon. The test line contains an anti-biotin antibody that "captures" the double-labeled amplicons. Since the double-labeled amplicon binds to the mobile reporter, it is colored with a reddish pink line in the case where the detection line is captured by the anti-biotin antibody. In another aspect, the positive control line comprises a secondary anti-FAM antibody that captures the reporter reporter, either not bound to the amplicon, or is bound to the anti-biotin antibody capture and expansion on the undetected line. Move the report. The positive control line is used to confirm that the mobile reporter has flowed through the membrane. The capture of the mobile reporter at the positive control line also produces a reddish red line. The overall LFD analysis was performed at room temperature.

RPA-LFD測定法之分子特異性Molecular specificity of RPA-LFD assay

RPA-LFD測定法的分子特異性使用該RPA P001、P002引子以及LF探針1,並經由交叉反應測試來評估,該反應使用從兩種其他動物病毒(即山羊痘病毒及牛白血病病毒)萃取的DNA或cDNA模板100ng,在已確定的偵測CAEV條件下進行。病毒標本由臺灣行政院農委會家畜衛生試驗所提供。該結果產物以LFD和4%AGE進行分析。 The molecular specificity of the RPA-LFD assay was evaluated using the RPA P001, P002 primer and LF probe 1, and was evaluated by cross-reactivity assay using two other animal viruses (ie, goat pox virus and bovine leukemia virus). 100 ng of the DNA or cDNA template was performed under established conditions for detection of CAEV. The virus specimens were provided by the Animal Health Laboratory of the Taiwan Executive Yuan. The resulting product was analyzed as LFD and 4% AGE.

使用LFD及AGE評估RPA分子靈敏度Evaluation of RPA molecular sensitivity using LFD and AGE

從感染CAEV的山羊萃取總DNA進行5倍連續稀釋50ng、10ng、2ng、400pg和80pg,以及該質體DNA進行10倍連續稀釋108、107、106、105、104、103、102和10倍拷貝數以作為RPA模板。通過LFD和AGE分析比較所得的DNA產物。 The total DNA was extracted from goats infected with CAEV for 5 times serial dilutions of 50 ng, 10 ng, 2 ng, 400 pg and 80 pg, and the plastid DNA was subjected to 10-fold serial dilutions 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 and 10 copy numbers are used as RPA templates. The resulting DNA product was compared by LFD and AGE analysis.

與血清學ELISA進行RPA-LFD的可信度和陽性率比較Comparison of credibility and positive rate of RPA-LFD with serological ELISA

同時使用RPA-LFD分析和血清學ELISA套組進行未知CAEV感染狀態的山羊樣本篩檢如後:(1)該RPA-LFD檢測根據先前所述使用基因組DNA之現場樣本診斷CAEV之步驟進行;(2)該血清學ELISA使用來自Bommeli Diagnostics公司(瑞士里貝菲爾德-伯恩)的Checkit MVV/CAEV全病毒ELISA,按照製造商說明書,以血清樣本進行測試。並且透過係數進行McNemar檢定一致性的計算,0等同沒有超過偶然一致性,而1.0則表示RPA-LFD與ELISA檢測之間具有完美一致性。 Simultaneous screening of goat samples with unknown CAEV infection status using RPA-LFD analysis and serological ELISA kits: (1) The RPA-LFD assay is performed according to the procedure for diagnosing CAEV using the on-site sample of genomic DNA as previously described; 2) The serological ELISA was tested with a serum sample using a Checkit MVV/CAEV whole virus ELISA from Bommeli Diagnostics (Ribery-Bern, Switzerland) according to the manufacturer's instructions. And the coefficient of McNemar test consistency was calculated by the coefficient. The 0 equivalent did not exceed the accidental consistency, while the 1.0 indicates the perfect consistency between the RPA-LFD and the ELISA test.

[結果][result]

RPA-LFD條件確定RPA-LFD condition determination

RPA反應的優選溫度是以103倍和102倍拷貝數的質體DNA 作為模板來確認,該拷貝數擴增分別在37℃、38℃或39℃下進行。由AGE測定的該擴增結果在37℃、38℃或39℃下,並未顯現出顯著擴增差異(數據未示)。因此,已知該檢測結果和該製造商的指引說明,選擇37℃作為分析溫度。使用102倍拷貝數的質體DNA作為模板在37℃下進行10、20、30、45和60分鐘的RPA反應,30、45和60分鐘的擴增產物量相似,但高於10和20分鐘的擴增產物量。事實上,由AGE測定的該擴增產物,在10分鐘時幾乎無法觀察到結果,且在20分鐘時僅能觀察到微弱結果(如圖1所示)。因此,為了在現場能實際且有效率的使用,選擇30分鐘培養時間作為優選條件。也使用LF試驗片檢測RPA擴增產物的偵測。經過短至10分鐘的培養時間即可偵測到該擴增產物,而30分鐘則可觀察到清晰的陽性檢測帶(如圖1)。 The preferred temperature for the RPA reaction is confirmed by using 10 3 fold and 10 2 copy number of plastid DNA as a template, and the copy number amplification is carried out at 37 ° C, 38 ° C or 39 ° C, respectively. The amplification results as determined by AGE did not show significant amplification differences at 37 ° C, 38 ° C or 39 ° C (data not shown). Therefore, the test results and the manufacturer's guidance instructions are known, and 37 ° C is selected as the analysis temperature. The RPA reaction at 10, 20, 30, 45, and 60 minutes was performed at 37 ° C using 10 2 copy number of plastid DNA as a template, and the amount of amplification products at 30, 45, and 60 minutes was similar, but higher than 10 and 20 The amount of amplification product in minutes. In fact, the amplification product determined by AGE showed almost no results at 10 minutes, and only weak results were observed at 20 minutes (as shown in Figure 1). Therefore, in order to be practical and efficient in use in the field, a 30 minute culture time is selected as a preferred condition. LF test strips were also used to detect the detection of RPA amplification products. The amplified product was detected by a culture time as short as 10 minutes, and a clear positive test band was observed in 30 minutes (Fig. 1).

RPA-LFD的分子特異性Molecular specificity of RPA-LFD

對於RPA P001、P002引子以及LF探針1在優選條件下之特異性檢測,該反應與來自其他脊椎動物病毒的DNA或cDNA一起進行。在與其他脊椎病毒如山羊痘病毒和牛白血病病毒以AGE(圖2A)和LFD(圖2B)一起檢驗和偵測的情況下,沒有交叉反應,表示該引子和該探針對於CAEV gag基因偵側具有特異性。然而,階梯狀(Ladder-like)圖形和引子/探針殘基偶爾會在AGE以非優選之條件下檢測時出現,例如:非優選之培養溫度、非優選之培養時間和非優選之模板DNA質和量。因此,也評估LFD分析法從非相關擴增子或引子/探針殘基中,辨別出其標靶擴增產物的能力。該LFD分析法僅對其期望的186-bp標靶擴增產物顯現陽性結果,即,在沒有模板控制(no template control,NTC)僅有以AGE確認的引子/探針殘基時,沒有出現偽陽性結果,並且僅顯現紅紫色的控制線(圖2B)。 For the specific detection of RPA P001, P002 primer and LF probe 1 under preferred conditions, the reaction is carried out with DNA or cDNA from other vertebrate viruses. In the case of testing and detection with other vertebrates such as goat pox virus and bovine leukemia virus with AGE (Fig. 2A) and LFD (Fig. 2B), there is no cross reaction, indicating that the primer and the probe are for the CAEV gag gene side. Specific. However, ladder-like patterns and primer/probe residues occasionally occur when AGE is detected under non-preferred conditions, such as: non-preferred culture temperature, non-preferred culture time, and non-preferred template DNA. Quality and quantity. Therefore, the ability of the LFD assay to discriminate its target amplification products from non-related amplicon or primer/probe residues was also evaluated. The LFD assay showed only positive results for its desired 186-bp target amplification product, ie, no primer/probe residues confirmed by AGE in no template control (NTC) False positive results, and only reddish purple control lines appear (Fig. 2B).

RPA-AGE分析和RPA-LFD分析的分子靈敏度比較Comparison of Molecular Sensitivity between RPA-AGE Analysis and RPA-LFD Analysis

在檢測該RPA-LFD分析敏感度的情況下,使用5倍序列稀釋的總DNA萃取物,發現山羊總DNA的80pg為偵測極限(圖3B)。此較RPA-AGE分析之偵側極限50ng更優625倍(圖3A)。在以重組質體作為模板的情況下,該RPA-LFD分析顯示偵測極限在10倍拷貝數有陽性訊號(圖4B),但該RPA-AGE僅在100倍拷貝數才顯現微弱帶狀(圖4A)。這些結果表明使用LFD法偵側RPA產物相較於使用AGE法偵側該RPA產物具有更高靈敏度,即使在以AGE檢測並不清晰的情況下,以LFD可產生更強的陽性訊號。 In the case of detecting the sensitivity of the RPA-LFD analysis, using a 5-fold serial dilution of the total DNA extract, it was found that 80 pg of total goat DNA was the detection limit (Fig. 3B). This is 625 times better than the detection limit of 50 ng for the RPA-AGE analysis (Fig. 3A). In the case of recombinant plastids as a template, the RPA-LFD analysis showed a positive signal at the 10-fold copy number of the detection limit (Fig. 4B), but the RPA-AGE showed a weak band only at 100-fold copy number (Fig. 4B). Figure 4A). These results indicate that using the LFD method to detect the RPA product is more sensitive than using the AGE method to detect the RPA product, and that LFD can produce a stronger positive signal even when the AGE detection is not clear.

與血清學ELISA進行RPA-LFD的可信度和陽性率比較Comparison of credibility and positive rate of RPA-LFD with serological ELISA

為了評估該RPA-LFD分析在統計學上的靈敏度和特異性,將其與傳統ELISA分析進行在場樣本的平行測試。該ELISA與該RPA-LFD分析結果的比較如表1所示。在以ELISA和RPA-LFD檢測的200個山羊樣本中,157隻(78.5%)在兩種分析下皆呈現陽性。在200隻山羊中,有43隻(21.5%)和36隻(18.0%)分別對ELISA和RPA-LFD分析呈現陰性,在43隻血清陰性的山羊中有7隻(3.5%)於RPA-LFD分析中呈現陽性。因此,該RPA-LFD分析(82%)相較於ELISA分析(78.5%)有顯著較高的CAEV陽性率(P<0.05)。係數為0.89,表示兩分析間有強烈的一致性。 To assess the statistical sensitivity and specificity of this RPA-LFD assay, it was tested in parallel with the on-site samples with conventional ELISA assays. The comparison of the ELISA with the results of the RPA-LFD analysis is shown in Table 1. Of the 200 goat samples tested by ELISA and RPA-LFD, 157 (78.5%) were positive for both analyses. Of the 200 goats, 43 (21.5%) and 36 (18.0%) were negative for ELISA and RPA-LFD analysis, respectively, and 7 (3.5%) of 43 seronegative goats were at RPA-LFD. Positive in the analysis. Therefore, the RPA-LFD analysis (82%) had a significantly higher CAEV positive rate (P < 0.05) compared to the ELISA analysis (78.5%). The coefficient is 0.89, indicating a strong consistency between the two analyses.

該LFD方法所展現較高的靈敏度和特異性是歸因於使用金標記之抗-FAM抗體以特異性捕捉FAM-標記的RPA擴增子,其具有在非優選擴增條件下,從非相關擴增子中辨別該標靶擴增子的能力。即使在該擴增產物量非常低,低至在AGE中幾乎無法顯現的情況下,該LFD方法仍能在試驗片上提供一可見的帶狀(圖3)。顯然該LFD分析擁有高靈敏度,在透過以RPA擴增子的特性辨識確認之下,能對抗偶然失配DNA標靶序列,同時也提供比AGE法更好的靈敏度。沒有擴增子和探針的雜交步驟,藉由LFD偵測RPA擴增子是簡單且可在室溫5-15分鐘測定。 The higher sensitivity and specificity exhibited by this LFD method is due to the use of gold-labeled anti-FAM antibodies to specifically capture FAM-labeled RPA amplicons, which have unrelated under non-preferred amplification conditions. The ability of the amplicon to discriminate the target amplicon. The LFD method provides a visible band on the test piece even when the amount of the amplified product is very low, as low as in the AGE (Fig. 3). Obviously, the LFD assay has high sensitivity and is able to counteract the accidental mismatched DNA target sequence, while also providing better sensitivity than the AGE method, by confirming the identity of the RPA amplicon. Without the hybridization step of the amplicon and probe, the detection of the RPA amplicon by LFD is simple and can be determined at room temperature for 5-15 minutes.

在本發明中,RPA法已經成功的發展用於擴增該CAEV的gag基因區,且該擴增子可以藉由該LF試驗片上半定量比色變化簡單地測定(圖4B)。在37℃下僅培養30分鐘即可檢測到擴增反應,因此,在流行地區,藉由使用加熱塊、水浴、電池供電裝置或甚至人體體溫進行培養即已足夠,去除了對外部電源和基礎設施的需求。本發明也顯示該RPA-LFD分析提供了相較於ELISA分析顯著較高的CAEV-陽性率(P<0.05),結果可能是由於RPA理論上甚至可以檢測到單一模板DNA拷貝數的事實。在臨床評估中,結果也顯示該RPA-LFD分析相較於正規的ELISA分析更為靈敏。這些結果也表明CAEV可在沒有可檢測之抗體存在的情況下,以藉由RPA-LFD分析測定。 In the present invention, the RPA method has been successfully developed for amplifying the gag gene region of the CAEV, and the amplicons can be simply determined by the above semi-quantitative colorimetric change of the LF test piece (Fig. 4B). The amplification reaction can be detected by culturing at 37 ° C for only 30 minutes. Therefore, in a popular area, it is sufficient to use a heating block, a water bath, a battery-powered device, or even a human body temperature to remove the external power supply and the foundation. The needs of the facility. The present invention also shows that the RPA-LFD analysis provides a significantly higher CAEV-positive rate (P < 0.05) compared to ELISA analysis, and the result may be due to the fact that RPA can theoretically even detect a single template DNA copy number. In the clinical evaluation, the results also showed that the RPA-LFD analysis was more sensitive than the regular ELISA analysis. These results also indicate that CAEV can be assayed by RPA-LFD analysis in the absence of detectable antibodies.

因此,本發明提供一種具靈敏度和特異性之RPA-LFD在場診斷方法,以簡單、快速且有成本效益的方法偵測CAEV原病毒DNA。RPA擴增和LFD檢測的結合,使總分析時間約為35分鐘,其僅為RPA-AGE分析 時間的四分之一,且比目前用於檢測CAEV的ELISA分析快了約6倍。高靈敏度和特異性、檢測時間短以及藉由側流層析雜合法確認該擴增子等,為本方法的關鍵優勢。步驟除一簡單加熱塊外不需其他設備,使CAEV感染的診斷在設備不足的實驗室或甚至在場進行變為可能。 Accordingly, the present invention provides a sensitivity and specificity of the RPA-LFD presence diagnostic method for detecting CAEV proviral DNA in a simple, rapid and cost effective manner. The combination of RPA amplification and LFD detection resulted in a total analysis time of approximately 35 minutes, which was only RPA-AGE analysis. A quarter of the time is about 6 times faster than the ELISA assay currently used to detect CAEV. High sensitivity and specificity, short detection time, and confirmation of the amplicon by lateral flow chromatography are the key advantages of this method. The procedure eliminates the need for additional equipment in addition to a simple heating block, making the diagnosis of CAEV infection possible in laboratories with insufficient equipment or even presence.

於此已揭示實施例,惟應瞭解其他變化的可能。此般變化並非遠離本發明之實施例範圍及精神,且所有對於本領域技藝人士顯而易見之修飾與變化,皆包含於如後之申請專利範圍之中。 Embodiments have been disclosed herein, but other variations are possible. The modifications and variations that are obvious to those skilled in the art are included in the scope of the appended claims.

<110> 國立臺灣大學 <110> National Taiwan University

<120> 在場檢測山羊關節炎腦炎病毒感染之方法及套組 <120> Methods and kits for detecting goat arthritis encephalitis virus infection in the field

<130> P161289TW <130> P161289TW

<160> 4 <160> 4

<170> PatentIn version 3.5 <170> PatentIn version 3.5

<210> 1 <210> 1

<211> 32 <211> 32

<212> DNA <212> DNA

<213> 未知 <213> Unknown

<220> <220>

<223> 正向引子 <223> Forward introduction

<220> <220>

<221> 引子_結合 <221> Introduction_Combination

<222> (1)..(32) <222> (1)..(32)

<400> 1 <400> 1

<210> 2 <210> 2

<211> 29 <211> 29

<212> DNA <212> DNA

<213> 未知 <213> Unknown

<220> <220>

<223> 反向引子 <223> Reverse primer

<220> <220>

<221> 引子_結合 <221> Introduction_Combination

<222> (1)..(29) <222> (1)..(29)

<400> 2 <400> 2

<210> 3 <210> 3

<211> 30 <211> 30

<212> DNA <212> DNA

<213> 未知 <213> Unknown

<220> <220>

<223> 探針 <223> Probe

<220> <220>

<221> misc_特徵 <221> misc_ feature

<222> (1)..(30) <222> (1)..(30)

<400> 3 <400> 3

<210> 4 <210> 4

<211> 15 <211> 15

<212> DNA <212> DNA

<213> 未知 <213> Unknown

<220> <220>

<223> 探針下游 <223> Downstream of the probe

<220> <220>

<221> misc_特徵 <221> misc_ feature

<222> (1)..(15) <222> (1)..(15)

<400> 4 <400> 4

Claims (8)

一種用以偵測山羊關節炎腦炎病毒(caprine arthritis-encephalitis virus,CAEV)感染之方法,其步驟包含:(a)於一對具有SEQ ID NO.1序列之正向引子和具有SEQ ID NO.2序列之反向引子以及一具有SEQ ID NO.3及SEQ ID NO.4之探針的存在下,透過重組酶聚合酶擴增(recombinase polymerase amplification,RPA)樣本中CAEV的gag段DNA之保守區(nt 618-803,186bp)以作為標靶序列,該探針係互補於該標靶序列的一內部區域,其中一引子具有一第一標記,該探針具有一第二標記,使該標靶序列擴增產生一具有第一和第二標記之擴增子;(b)將步驟(a)的擴增產物滴加至一側流層析裝置/試驗片(lateral flow device/strip),以側向流至該側流層析裝置/試驗片的遠端,其中該側流層析裝置/試驗片依序包含(i)一樣品墊、(ii)一接合墊、(iii)一檢測區以及(iv)一控制區;所述接合墊具有以第一作用物標記之移動報告子,該第一作用物可特異性地結合至該擴增子的該第一標記或該第二標記;所述檢測區具有一第二作用物,其特異性地結合該擴增子的該第二標記或該第一標記,以抑制與移動報告子結合的該擴增子進一步地側流層析;所述控制區提供一控制作用物;及(c)偵測在該檢測區是否有任何擴增產物的結合。 A method for detecting a caprine arthritis-encephalitis virus (CAEV) infection, the method comprising the steps of: (a) a pair of a forward primer having the sequence of SEQ ID NO. 1 and having SEQ ID NO a reverse primer of the sequence of 2 and a gag segment DNA of CAEV in a recombinase polymerase amplification (RPA) sample in the presence of a probe having SEQ ID NO. 3 and SEQ ID NO. a conserved region (nt 618-803, 186 bp) as a target sequence, the probe is complementary to an internal region of the target sequence, wherein an primer has a first label and the probe has a second label The target sequence is amplified to produce an amplicon having first and second labels; (b) the amplification product of step (a) is added dropwise to a lateral flow device/strip Flowing laterally to the distal end of the lateral flow chromatography device/test piece, wherein the lateral flow chromatography device/test piece sequentially comprises (i) a sample pad, (ii) a bonding pad, (iii) a a detection zone and (iv) a control zone; the bonding pad having a movement reporter marked with a first substrate, the first The substance may specifically bind to the first label or the second label of the amplicon; the detection region has a second substrate that specifically binds to the second label or the first of the amplicon a marker for inhibiting further amplification of the amplicons associated with the mobile reporter; the control region provides a control substrate; and (c) detecting whether there is any binding of the amplification product in the detection region . 如請求項1所述之方法,其中該第一和第二標記是選自於生物素(biotin)、螢光素衍生物(fluorescein derivatives)、玫瑰紅衍生物(rhodamine derivatives)、級聯藍(Cascade Blue)、螢光黃(Lucifer yellow)、5-溴-2-去 氧尿苷(5-bromo-2-deoxyuridine,BrdU)、二硝基酚(dinitrophenol,DNP)、地谷新配質(digoxygenin,DIG)、及短肽標記序列。 The method of claim 1, wherein the first and second markers are selected from the group consisting of biotin, fluorescein derivatives, rhodamine derivatives, and cascade blue ( Cascade Blue), Lucifer yellow, 5-bromo-2-de 5-bromo-2-deoxyuridine (BrdU), dinitrophenol (DNP), digoxygenin (DIG), and short peptide labeling sequences. 如請求項2所述之方法,其中該反向引子具有一5’端生物素(5’-biotin)標記。 The method of claim 2, wherein the reverse primer has a 5'-biotin label. 如請求項3所述之方法,其中該探針係由具有5’端FAM(5’-FAM)抗原標記的SEQ ID NO.3,且以THF間隔子(THF spacer)連接至一在3’終端帶有一C3間隔子(C3-spacer)之SEQ ID NO.4序列下游所組成。 The method of claim 3, wherein the probe is SEQ ID NO. 3 having a 5'-end FAM (5'-FAM) antigen marker and is ligated to a 3' with a THF spacer (THF spacer) The terminal consists of a downstream sequence of SEQ ID NO. 4 with a C3 spacer (C3-spacer). 如請求項4所述之方法,其中該移動報告子係一種金、二氧化矽(silica)、硒、聚苯乙烯(polystyrene)、三聚氰胺樹脂(melamine resin)、聚甲基丙烯酸鹽(polymethacrylate)、苯乙烯/二乙烯苯(styrene/divinylbenzene)共聚物或是聚乙烯基甲苯(polyvinyltoluene)之微粒。 The method of claim 4, wherein the mobile reporter is a gold, silica, selenium, polystyrene, melamine resin, polymethacrylate, A styrene/divinylbenzene copolymer or a particle of polyvinyltoluene. 一種用於偵側樣本中山羊關節炎腦炎病毒(CAEV)之套組,其包含:一對具有SEQ ID NO.1序列之正向引子和具有SEQ ID NO.2序列之反向引子,以CAEV gag段(nt 512-1858)DNA作為標靶序列為目標;一探針,其互補於該標靶序列的一內部區域,具有SEQ ID NO.3及SEQ ID NO.4;一反應劑,用以進行重組酶聚合酶擴增反應;以及一側流層析裝置/試驗片;其中一引子具有一第一標記,該探針具有一第二標記,使該標靶序列擴增產生一標記有第一和第二標記之擴增子;且該側流層析裝置/試驗片依序包含(i)一樣品墊、(ii)一接合墊、(iii)一檢測區以及(iv)一控制區,所述接合墊具有以第一作用物標記之移動報告子,該第一作用物特異性地結 合該擴增子的該第一標記或該第二標記,所述檢測區具有一第二作用物,其特異性地結合該第二標記或該第一標記,所述控制區提供一控制作用物。 A kit for detecting a goat arthritis encephalitis virus (CAEV) in a side sample comprising: a pair of a forward primer having the sequence of SEQ ID NO. 1 and a reverse primer having the sequence of SEQ ID NO. 2, The CAEV gag segment (nt 512-1858) DNA targets the target sequence; a probe complementary to an internal region of the target sequence, having SEQ ID NO. 3 and SEQ ID NO. 4; a reagent, For performing a recombinase polymerase amplification reaction; and a one-stream chromatography device/test piece; wherein the primer has a first label, the probe has a second label, and the target sequence is amplified to generate a label a first and a second labeled amplicons; and the lateral flow chromatography device/test piece sequentially comprises (i) a sample pad, (ii) a bonding pad, (iii) a detection zone, and (iv) a a control zone, the bonding pad having a movement reporter labeled with a first substrate, the first substrate specifically binding Combining the first marker or the second marker of the amplicon, the detection zone has a second substrate that specifically binds the second marker or the first marker, and the control region provides a control function Things. 如請求項6所述之套組,其進一步包含一反應劑,用以從該樣品中萃取核酸。 The kit of claim 6 further comprising a reagent for extracting nucleic acid from the sample. 如請求項6所述之套組,其中該反向引子具有一5’端生物素(5’-biotin)標記,且該探針係由具有5’端FAM(5’-FAM)抗原標記的SEQ ID NO.3,且以THF間隔子(THF spacer)連接至一在3’終端帶有一C3間隔子之SEQ ID NO.4序列下游所組成,該移動報告子係一標記有一抗FAM(anti-FAM)抗體之微粒,且該控制作用物係一該抗FAM(anti-FAM)抗體之次級抗體。 The kit of claim 6, wherein the reverse primer has a 5'-biotin label and the probe is labeled with a 5'-end FAM (5'-FAM) antigen SEQ ID NO. 3, and is ligated with a THF spacer (THF spacer) to a downstream of the sequence of SEQ ID NO. 4 with a C3 spacer at the 3' terminus, the mobile reporter is labeled with an anti-FAM (anti -FAM) microparticles of the antibody, and the control substrate is a secondary antibody against the FAM (anti-FAM) antibody.
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