TWI765501B - Primer pair, method and detecting device for detecting colletotrichum siamense - Google Patents

Primer pair, method and detecting device for detecting colletotrichum siamense Download PDF

Info

Publication number
TWI765501B
TWI765501B TW109146642A TW109146642A TWI765501B TW I765501 B TWI765501 B TW I765501B TW 109146642 A TW109146642 A TW 109146642A TW 109146642 A TW109146642 A TW 109146642A TW I765501 B TWI765501 B TW I765501B
Authority
TW
Taiwan
Prior art keywords
primer pair
seq
polymerase chain
chain reaction
strawberry
Prior art date
Application number
TW109146642A
Other languages
Chinese (zh)
Other versions
TW202225413A (en
Inventor
曹嘉惠
鄭光琪
呂長益
鐘珮哲
吳竑毅
Original Assignee
財團法人工業技術研究院
行政院農業委員會苗栗區農業改良場
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 財團法人工業技術研究院, 行政院農業委員會苗栗區農業改良場 filed Critical 財團法人工業技術研究院
Priority to TW109146642A priority Critical patent/TWI765501B/en
Application granted granted Critical
Publication of TWI765501B publication Critical patent/TWI765501B/en
Publication of TW202225413A publication Critical patent/TW202225413A/en

Links

Images

Landscapes

  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The present disclosure provides primer pairs, method and detecting device for detecting Colletotrichum siamense. The method includes the following steps: (a) obtaining a strawberry DNA sample to be tested; (b) performing a polymerase chain reaction on the strawberry DNA sample with a specific primer pair to obtain a polymerase chain reaction product, and (c) detecting whether the polymerase chain reaction product has a DNA gene of Colletotrichum siamense.

Description

用於檢測草莓炭疽病的方法、引子對、以及檢測裝置Method, primer pair, and detection device for detecting strawberry anthracnose

本揭露係關於一種用於檢測草莓炭疽病的方法、引子對、以及檢測裝置,具體而言,關於一種利用等溫聚合酶連鎖反應針對草莓炭疽菌( Colletotrichum siamense)進行檢測之方法、引子對、以及檢測裝置。 The present disclosure relates to a method, primer pair, and detection device for detecting strawberry anthracnose , in particular, to a method, primer pair, and detection device.

草莓( Fragaria ananassaDuch.)是薔薇科草莓屬植物中最常見的一雜交種,屬於重要的高經濟作物。然而,由於草莓的育苗期間長達6個月以上,且會橫跨高溫多濕的夏季,因此種苗極易受到病害之感染,其中又以炭疽病最為嚴重。 Strawberry ( Fragaria ananassa Duch.) is one of the most common hybrids in the Rosaceae Strawberry genus, and is an important high-economic crop. However, since strawberry seedlings are raised for more than 6 months and will span the hot and humid summer, the seedlings are very susceptible to diseases, among which anthracnose is the most serious.

草莓炭疽病好發於草莓苗數量快速增加時期,因而極易造成育苗農民損失。再者,由於炭疽病菌可能潛伏感染於植株中,因此即使度過育苗期,定植後仍有可能因高溫或未良好防治,而有大量植株死亡的情況,因此在培育草莓的過程中,定期監控植株是否帶有炭疽病菌為重要的防治措施。Strawberry anthracnose occurs during the period of rapid increase in the number of strawberry seedlings, which can easily cause losses to seedling farmers. Furthermore, since anthracnose may be latently infected in plants, even after the seedling period, a large number of plants may die due to high temperature or poor control after planting. Therefore, in the process of cultivating strawberries, regular monitoring Whether the plant has anthracnose is an important control measure.

造成草莓炭疽病的菌種甚多,其中草莓炭疽菌( Colletotrichum siamense)為台灣特有種,其為在台灣地區造成草莓感染到炭疽病最為常見的菌種,因而對台灣的草莓產量的影響甚劇。然而,現今僅能以聚合酶連鎖反應(PCR)針對草莓炭疽病進行檢測,因而檢測步驟繁複,通常需要將檢體攜回檢驗實驗室才能完成檢測,故相當不利於田野調查。再者,用於檢測草莓炭疽菌的檢測方法所針對的菌種多為似膠黏孢炭疽刺盤孢菌( Colletotrichum gloeosporioides)等的病原菌,因而以習知的檢測試劑進行檢測時,仍有準確率不足之問題。 There are many bacterial species that cause strawberry anthracnose, among which Colletotrichum siamense is endemic to Taiwan. It is the most common bacterial species that causes strawberry to be infected with anthracnose in Taiwan, so it has a great impact on Taiwan's strawberry yield. . However, currently, only the polymerase chain reaction (PCR) can be used to detect strawberry anthracnose, so the detection steps are complicated, and the samples usually need to be brought back to the testing laboratory to complete the detection, which is quite unfavorable for field investigations. In addition, the detection methods used for the detection of strawberry anthracnose are mostly pathogenic bacteria such as Colletotrichum gloeosporioides , so when the conventional detection reagents are used for detection, it is still accurate. The problem of insufficient accuracy.

因此,目前仍亟需一種草莓炭疽菌的檢測方法,以期望能方便、縮短檢測時間並能夠具有優異準確率之新穎檢測方法。Therefore, there is still an urgent need for a detection method for strawberry anthracnose, and a novel detection method that is convenient, shortens detection time and can have excellent accuracy is expected.

本揭露提供一種用於檢測草莓炭疽病( Colletotrichum siamense)的方法,其包括以下步驟:(a)獲得一待測草莓之DNA樣本;(b)將該待測草莓之DNA樣本作為模版以一特定引子對進行聚合酶連鎖反應,以獲得一聚合酶連鎖反應產物;以及(c)檢測該聚合酶連鎖反應產物中是否具有一草莓炭疽菌的DNA基因,其中該特定引子為包含序列辨識號:2至序列辨識號:5的第一引子對、包含序列辨識號:10至序列辨識號:13的第二引子對、序列辨識號:18及19的第三引子對、序列辨識號:20及21的第四引子對、或序列辨識號:22及23的第五引子對。 The present disclosure provides a method for detecting Colletotrichum siamense , comprising the following steps: (a) obtaining a DNA sample of the strawberry to be tested; (b) using the DNA sample of the strawberry to be tested as a template with a specific performing a polymerase chain reaction on the primer pair to obtain a polymerase chain reaction product; and (c) detecting whether the polymerase chain reaction product has a DNA gene of Anthracis fragariae, wherein the specific primer is a sequence identification number: 2 First primer pair to SEQ ID: 5, second primer pair including SEQ ID: 10 to SEQ ID: 13, third primer pair to SEQ ID: 18 and 19, SEQ ID: 20 and 21 The fourth primer pair of , or the fifth primer pair of SEQ ID NOs: 22 and 23.

本揭露另提供一種用於檢測草莓炭疽病的引子對,其包括序列辨識號:2至序列辨識號:5的引子對。The present disclosure further provides a primer pair for detecting strawberry anthracnose, which includes a primer pair of SEQ ID NO: 2 to SEQ ID NO: 5.

本揭露另提供一種用於檢測草莓炭疽病的引子對,其包括序列辨識號:10至序列辨識號:13的引子對。The present disclosure further provides a primer pair for detecting strawberry anthracnose, which includes a primer pair of SEQ ID NO: 10 to SEQ ID NO: 13.

本揭露另提供一種用於檢測草莓炭疽病的引子對,其為序列辨識號:18至序列辨識號:19的引子對。The present disclosure further provides a primer pair for detecting strawberry anthracnose, which is a primer pair of SEQ ID NO: 18 to SEQ ID NO: 19.

本揭露另提供一種用於檢測草莓炭疽病的引子對,其為序列辨識號:20至序列辨識號:21的引子對。The present disclosure further provides a primer pair for detecting strawberry anthracnose, which is a primer pair of SEQ ID NO: 20 to SEQ ID NO: 21.

本揭露另提供一種用於檢測草莓炭疽病的引子對,其為序列辨識號:22至序列辨識號:23的引子對。The present disclosure further provides a primer pair for detecting strawberry anthracnose, which is a primer pair of SEQ ID NO: 22 to SEQ ID NO: 23.

本揭露另提供一種用於檢測草莓炭疽病的檢測裝置,其包括:一控溫模組,其使用一模糊控制程式及一比例積分微分(PID)控制程式進行調控;以及一恆溫槽,其與該控溫模組電性連接,該恆溫槽中設置有至少一個反應槽,其中將一待測草莓之DNA樣本作為模版與一特定引子對置入該反應槽,以在一特定溫度下進行聚合酶連鎖反應,其中該特定引子對係為上述中任一之引子對。The present disclosure further provides a detection device for detecting strawberry anthracnose, which includes: a temperature control module, which is regulated by a fuzzy control program and a proportional integral derivative (PID) control program; and a constant temperature tank, which is connected with The temperature control module is electrically connected, and at least one reaction tank is set in the constant temperature tank, wherein a DNA sample of strawberry to be tested is used as a template and a specific primer pair is placed in the reaction tank to conduct polymerization at a specific temperature Enzyme chain reaction, wherein the specific primer pair is any one of the primer pairs described above.

為了讓本揭露之上述和其他目的、特徵、和優點能更明顯易懂,下文特舉實施例,並配合所附圖示,其詳細說明如下:In order to make the above-mentioned and other objects, features, and advantages of the present disclosure more obvious and easy to understand, the following specific embodiments are given in conjunction with the accompanying drawings, and the detailed descriptions are as follows:

本揭露可藉由下述實施方式中所揭示之各種揭露態樣、實施例及表列之相關敘述所瞭解。除非在本文中另作定義,否則與本揭露關聯使用之術語(包含技術及科學術語)應具有本揭露所屬技術領域中具有通常知識者所瞭解之含義。且當可瞭解,除非本文中提供之定義另作說明,在任何潛在歧義之情況,術語之定義應與該等普遍使用之術語(如詞典中所定義)一致。可進一步瞭解者,本案所使用的術語僅係用作描述特定實施態樣之目的,而非用於限定。The present disclosure can be understood through the related descriptions of various disclosure aspects, examples and tables disclosed in the following embodiments. Unless otherwise defined herein, terms (including technical and scientific terms) used in connection with this disclosure shall have the meanings that are understood by those of ordinary skill in the art to which this disclosure belongs. And it should be understood that, in the event of any potential ambiguity, the definitions of terms shall be accorded to those commonly used terms (as defined in the dictionary) unless the definitions provided herein indicate otherwise. It can be further understood that the terms used in this application are only used for the purpose of describing specific implementation aspects, rather than being used for limitation.

必須注意的是,除非有清楚的相反指示,於說明書或申請專利範圍使用之單數格式「一」、「一種」及「該」亦包含複數表示。因此,除非上下文另有需要,單數術語應包含複數而複數術語亦包含單數。It must be noted that, unless clearly indicated to the contrary, the singular forms "a", "an" and "the" used in the specification or the scope of the patent application also include plural expressions. Accordingly, unless otherwise required by context, singular terms shall include pluralities and plural terms shall also include the singular.

以下就本揭露之檢測草莓炭疽病的方法、引子對、以及檢測裝置予以詳盡說明。The method, primer pair, and detection device for detecting strawberry anthracnose disclosed in the present disclosure will be described in detail below.

如本文中所使用,術語「聚合酶連鎖反應(polymerase chain reaction, PCR)」係在未特別指定的情況下,係指任何用於產生許多特定DNA序列複製片段之生化技術。As used herein, the term "polymerase chain reaction (PCR)", unless otherwise specified, refers to any biochemical technique used to generate a number of replicated fragments of a particular DNA sequence.

如本文中所使用,術語「引子」(primer)係指能夠與核酸雜合且在適當條件下,在適當緩衝液中及在適合之溫度下,作為核苷酸(RNA或DNA)聚合之起始位點的寡核苷酸。引子之適當長度視引子之預期用途而定,但引子通常為至少7個核苷酸長,且長度更通常介於10至30個核苷酸之範圍內,或甚至更通常為15至30個核苷酸。然而,在其他特定情況下,其他引子可略較長,例如30至70個或40至60個核苷酸長。所屬技術領域中具有通常知識者瞭解,引子長度可處於由任何此等值限定的任何範圍內(例如7至70個或50至70個)。如本文進一步所述之各種長度的寡核苷酸可作為引子用於擴增等反應。As used herein, the term "primer" refers to a nucleic acid capable of hybridizing and under appropriate conditions, in an appropriate buffer, and at an appropriate temperature, as a starter for the polymerization of nucleotides (RNA or DNA) oligonucleotide at the initiation site. Appropriate lengths of primers depend on the intended use of the primer, but primers are generally at least 7 nucleotides in length, and more typically range from 10 to 30 nucleotides in length, or even more typically 15 to 30 nucleotides in length Nucleotides. However, in other specific cases, other primers may be somewhat longer, eg, 30 to 70 or 40 to 60 nucleotides in length. One of ordinary skill in the art understands that primer lengths can be in any range defined by any such value (eg, 7 to 70 or 50 to 70). Oligonucleotides of various lengths as further described herein can be used as primers for reactions such as amplification.

本文使用的,術語「引子對」(primer pair)是指一組或一對引子,其包含與待擴增序列之5’端上游序列(upstream sequence)互補且雜合的「正向引子」(forward primer)以及與待擴增序列之3’端下游序列(downstream sequence)互補並雜合的「反向引子」(reverse primer),然而本揭露的引子對不限於此,例如在等溫聚合酶連鎖反應的情況下,亦可舉出例如「內引子」(inner primer)、「外引子」(outer primer)等。所屬技術領域中具有通常知識者應理解,本文使用之術語「正向引子」等並非用以限制該引子,而僅是提供示例性的方向。As used herein, the term "primer pair" refers to a set or pair of primers comprising a "forward primer" that is complementary and hybrid to the 5' upstream sequence of the sequence to be amplified ( forward primer) and a "reverse primer" that is complementary to and hybridizes to the downstream sequence at the 3' end of the sequence to be amplified, but the primer pair of the present disclosure is not limited to this, for example, in isothermal polymerase In the case of a chain reaction, for example, an "inner primer", an "outer primer" and the like can also be mentioned. It should be understood by those of ordinary skill in the art that the terms "forward primer" and the like used herein are not intended to limit the primer, but merely provide an exemplary direction.

本文使用的「模版」(template)一詞是指待擴增的任何類型之核酸分子,較佳為待擴增的DNA序列,亦可稱為DNA模版。本揭示內容的「核酸模版」或「DNA模版」至少包含部分或完整的單股核酸序列或單股DNA序列。在對核酸模版進行的雜合反應中,所得到的複合物可包括單股分子和雙股分子。The term "template" as used herein refers to any type of nucleic acid molecule to be amplified, preferably a DNA sequence to be amplified, also referred to as a DNA template. A "nucleic acid template" or "DNA template" of the present disclosure comprises at least a partial or complete single-stranded nucleic acid sequence or single-stranded DNA sequence. In hybridization reactions performed on nucleic acid templates, the resulting complexes can include single-stranded and double-stranded molecules.

有鑑於上述目的,本揭露提供一種主要針對台灣特有種之草莓炭疽菌( Colletotrichum siamense)的檢測方法,其中該草莓炭疽菌之DNA包含序列辨識號如1所示的序列。 In view of the above-mentioned purpose, the present disclosure provides a detection method for Colletotrichum siamense , a species endemic to Taiwan, wherein the DNA of Colletotrichum siamense comprises the sequence as shown in SEQ ID NO: 1.

本揭露亦提出一種用於檢測草莓炭疽病的引子對,發明人針對上述揭示的序列辨識號:1序列設計一系列專一性引子對,以提高針對台灣特有種之草莓炭疽菌之檢測方法之準確度及檢測效率。The present disclosure also proposes a primer pair for detecting strawberry anthracnose. The inventors designed a series of specific primer pairs for the above disclosed SEQ ID NO: 1 to improve the accuracy of the detection method for strawberry anthracnose endemic to Taiwan. degree and detection efficiency.

本揭露更提出一種用於檢測草莓炭疽病的檢測裝置,藉由優異的控溫模組,藉以可以專一性地擴增台灣特有種之草莓炭疽菌之基因片段,因而能夠快速準確地獲得草莓炭疽病的檢測結果。The present disclosure further proposes a detection device for detecting strawberry anthracnose. With an excellent temperature control module, the gene fragment of strawberry anthracnose, which is endemic to Taiwan, can be specifically amplified, so that strawberry anthracnose can be obtained quickly and accurately. disease test results.

本揭露之一態樣提供一種用於草莓炭疽病的檢測方法。該方法主要是利用一待測草莓之DNA樣本,來檢測該待測草莓是否感染草莓炭疽病。本揭露之方法包含以下步驟:(a)獲得一待測草莓之DNA樣本;(b)將待測草莓之DNA樣本作為模版以一特定引子對進行聚合酶連鎖反應,以獲得聚合酶連鎖反應產物;以及(c)檢測該聚合酶連鎖反應產物中是否具有一草莓炭疽菌的DNA基因。如下文表1所示,該特定引子可為包含序列辨識號:2至序列辨識號:5的第一引子對、包含序列辨識號:10至序列辨識號:13的第二引子對、序列辨識號:18及19的第三引子對、序列辨識號:20及21的第四引子對、或序列辨識號:22及23的第五引子對。An aspect of the present disclosure provides a detection method for strawberry anthracnose. The method mainly uses a DNA sample of a strawberry to be tested to detect whether the strawberry to be tested is infected with strawberry anthracnose. The method of the present disclosure includes the following steps: (a) obtaining a DNA sample of the strawberry to be tested; (b) using the DNA sample of the strawberry to be tested as a template to perform a polymerase chain reaction with a specific primer pair to obtain a polymerase chain reaction product and (c) detecting whether the polymerase chain reaction product has a DNA gene of Strawberry anthracis. As shown in Table 1 below, the specific primers can be the first primer pair comprising SEQ ID NO: 2 to SEQ ID NO: 5, the second primer pair comprising SEQ ID NO: 10 to SEQ ID NO: 13, and the SEQ ID NO: 13 Numbers: 18 and 19, the third primer pair, SEQ ID numbers: 20 and 21, the fourth primer pair, or SEQ ID numbers: 22 and 23, the fifth primer pair.

[表1] 第一引子對 序列辨識號 引子名稱 序列 2 L1-F3 ATTGTCGACAATCGGACGG 3 L1-B3 CACGAACCTGGTAATCAGCT 4 L1-FIP GAAGTGTTAGTCGCCCCGCGTTTTTTACGGTTCCAATTGGGGTTTG 5 L1-BIP TGGTGATGCTTGAGACTTCCCGTTTTTTCCGGAGTCTAAGCAGATCCA 6 L1-F1c GAAGTGTTAGTCGCCCCGCG 7 L1-B1c TGGTGATGCTTGAGACTTCCCG 8 L1-F2 ACGGTTCCAATTGGGGTTTG 9 L1-B2 CCGGAGTCTAAGCAGATCCA 第二引子對 序列辨識號 引子名稱 序列 10 L2-F3 TCGACAATCGGACGGGAC 11 L2-B3 CCTCAACACGAACCTGGTAA 12 L2-FIP TCGGGAAGTCTCAAGCATCACCTTTTTTAGAAGGAAGAGCGGGGAAC 13 L2-BIP AATCCTAAGCACCTCACCGGCTTTTTTAGCTTTAAACAGCCGGCATT 14 L2-F1c TCGGGAAGTCTCAAGCATCACC 15 L2-B1c AATCCTAAGCACCTCACCGGC 16 L2-F2 AGAAGGAAGAGCGGGGAAC 17 L2-B2 AGCTTTAAACAGCCGGCATT 第三引子對 序列辨識號 引子名稱 序列 18 HP2-F ACAAACGGTGATCCTTTCGTCAGAAAC 19 HP2-R CGGTTGGGAGACAGCTCTTCTTTAACA 第四引子對 序列辨識號 引子名稱 序列 20 HI2-F TATGGTAACCACTGATCCCAAAG 21 HI2-R GCCTAAGGGATGTTTCTTAATGATATG 第五引子對 序列辨識號 引子名稱 序列 22 HI3-F GGTGATCCTTTCGTCAGAAACT 23 HI3-R TTGGGAGACAGCTCTTCTTTAAC 其他引子對 序列辨識號 引子名稱 序列 24 HP1-F CCTTTCGTCAGAAACTACGTTCCGTGT 25 HP1-R AGATTATCCGGTTGGGAGACAGCTCTT 26 HP3-F CCTTTCGTCAGAAACTACGTTCCGTGT 27 HP3-R CGGTTGGGAGACAGCTCTTCTTTAACA 28 HI1-F GAAGGAAGAGAAGAAGGCAGTAG 29 HI1-R CATTAGACCCTCGACGAATAGAAA [Table 1] first primer pair Serial identification number Introductory name sequence 2 L1-F3 ATTGTCGACAATCGGACGG 3 L1-B3 CACGAACCTGGTAATCAGCT 4 L1-FIP GAAGTGTTAGTCGCCCCGCGTTTTTTACGGTTCCAATTGGGGTTTG 5 L1-BIP TGGTGATGCTTGAGACTTCCCGTTTTTTCCGGAGTCTAAGCAGATCCA 6 L1-F1c GAAGTGTTAGTCGCCCCGCG 7 L1-B1c TGGTGATGCTTGAGACTTCCCG 8 L1-F2 ACGGTTCCAATTGGGGTTTG 9 L1-B2 CCGGAGTCTAAGCAGATCCA second primer pair Serial identification number Introductory name sequence 10 L2-F3 TCGACAATCGGACGGGAC 11 L2-B3 CCTCAACACGAACCTGGTAA 12 L2-FIP TCGGGAAGTCTCAAGCATCACCTTTTTTTAGAAGGAAGAGCGGGGAAC 13 L2-BIP AATCCTAAGCACCTCACCGGCTTTTTTAGCTTTAAACAGCCGGCATT 14 L2-F1c TCGGGAAGTCTCAAGCATCACC 15 L2-B1c AATCCTAAGCACCTCACCGGC 16 L2-F2 AGAAGGAAGAGCGGGGAAC 17 L2-B2 AGCTTTAAACAGCCGGCATT third primer pair Serial identification number Introductory name sequence 18 HP2-F ACAAACGGTGATCCTTTCGTCAGAAAC 19 HP2-R CGGTTGGGAGACAGCTCTTCTTTAACA fourth primer pair Serial identification number Introductory name sequence 20 HI2-F TATGGTAACCACTGATCCCAAAG twenty one HI2-R GCCTAAGGGATGTTTCTTAATGATATG fifth primer pair Serial identification number Introductory name sequence twenty two HI3-F GGTGATCCTTTCGTCAGAAACT twenty three HI3-R TTGGGAGACAAGCTCTTCTTTAAC other primer pairs Serial identification number Introductory name sequence twenty four HP1-F CCTTTCGTCAGAAACTACGTTCCGTGT 25 HP1-R AGATTATCCGGTTGGGAGACAGCTCTT 26 HP3-F CCTTTCGTCAGAAACTACGTTCCGTGT 27 HP3-R CGGTTGGGAGACAGCTCTTCTTTAACA 28 HI1-F GAAGGAAGAGAAGAAGGCAGTAG 29 HI1-R CATTAGACCCTCGACGAATAGAAA

在一特定實施例中,首先須從一待檢測草莓取得其DNA樣本(步驟(a))。依據另一特定實施方式,是從一疑似感染草莓炭疽病的草莓取得植物樣本。接著,可利用任何本領域具有通常知識者熟知的方法從所取得的植物樣本中(例如草莓葉、草莓果肉等)萃取該待檢測草莓的基因體DNA(genomic DNA)。萃取DNA的方法可包含首先利用物理作用、化學作用或溫差方式使細胞壁裂解;接著加入有機溶劑以純化細胞內的核酸。前述物理作用實例包含但不限於,超聲波震盪或研磨。化學作用包含利用裂解緩衝液來裂解細胞壁。前述溫差方式包括將細胞與液態氮混合並進行研磨,藉以使細胞壁破裂。接著,可添加乙醇或異丙醇來純化從細胞中釋放出的核酸。另一方面,也可以藉由商業化套組來達到相同的DNA萃取目的。該方法可由熟習此項技術者修改以自植物樣本中萃取DNA。In a specific embodiment, a DNA sample must first be obtained from a strawberry to be tested (step (a)). According to another specific embodiment, a plant sample is obtained from a strawberry suspected of being infected with strawberry anthracnose. Next, the genomic DNA (genomic DNA) of the strawberry to be tested can be extracted from the obtained plant samples (eg, strawberry leaves, strawberry pulp, etc.) by any method well known to those with ordinary knowledge in the art. The method of extracting DNA may include first lysing the cell wall by physical action, chemical action or temperature difference; then adding an organic solvent to purify the nucleic acid in the cell. Examples of the foregoing physical actions include, but are not limited to, ultrasonication or grinding. The chemical action involves the use of a lysis buffer to lyse the cell wall. The aforementioned thermal approach involves mixing the cells with liquid nitrogen and grinding, thereby disrupting the cell walls. Next, ethanol or isopropanol can be added to purify the nucleic acids released from the cells. On the other hand, commercial kits can also be used to achieve the same purpose of DNA extraction. This method can be modified by those skilled in the art to extract DNA from plant samples.

接著,在步驟(b)中,以自上述步驟獲得的待測草莓之DNA樣本作為模版,以一特定引子對進行聚合酶連鎖反應,以獲得聚合酶連鎖反應產物。依據一特定實施方式,特定引子可為包含序列辨識號:2至5的第一引子對、包含序列辨識號:10至13的第二引子對、序列辨識號:18及19的第三引子對、序列辨識號:20及21的第四引子對、或序列辨識號:22及23的第五引子對。Next, in step (b), using the DNA sample of the strawberry to be tested obtained from the above step as a template, a polymerase chain reaction is performed with a specific primer pair to obtain a polymerase chain reaction product. According to a specific embodiment, the specific primers can be the first primer pair comprising SEQ ID: 2 to 5, the second primer pair comprising SEQ ID: 10 to 13, the third primer pair comprising SEQ ID: 18 and 19 , the fourth primer pair with sequence identification numbers: 20 and 21, or the fifth primer pair with sequence identification numbers: 22 and 23.

具體而言,上述聚合酶連鎖反應的反應溫度可為約60至97℃。詳言之,反應溫度可例如為兩階段的溫度進行,但本揭露不限於此,該聚合酶連鎖反應亦可以在一恆定溫度下進行。當聚合酶連鎖反應的溫度以兩階段進行的情況下,其第一階段溫度可為約90至97℃,例如可為約91、92、93、94、95、96℃;而第二階段溫度可為約60~75℃,例如可為約61、62、63、64、65、66、67、68、69、70、71、72、73、74℃等,但不限於此。在另一特定實施方式,反應溫度為一階段進行的情況下,該反應溫度可為60~75℃,其例示的溫度可如上述兩階段中的第二階段溫度所示。Specifically, the reaction temperature of the above-mentioned polymerase chain reaction may be about 60 to 97°C. Specifically, the reaction temperature can be, for example, a two-stage temperature, but the present disclosure is not limited thereto, and the polymerase chain reaction can also be performed at a constant temperature. When the temperature of the polymerase chain reaction is carried out in two stages, the temperature of the first stage can be about 90 to 97°C, for example, it can be about 91, 92, 93, 94, 95, 96°C; and the temperature of the second stage It can be about 60~75°C, for example, about 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74°C, etc., but not limited thereto. In another specific embodiment, when the reaction temperature is carried out in one stage, the reaction temperature may be 60-75° C., and the exemplified temperature may be shown as the temperature of the second stage in the above two stages.

依據一特定實施方式,當聚合酶連鎖反應的溫度以兩階段進行的情況下,其第一階段的反應時間可為約1至5分鐘,例如可為約2、3、4分鐘;第二階段的反應時間可為約40至150分鐘,例如可為約45、50、55、60、65、70、75、80、85、90、95、100、105、110、115、120、125、130、135、140、145分鐘,但不限於此。然而,在另一特定實施方式,當聚合酶連鎖反應的溫度以一階段進行的情況下,其反應時間可以上述兩階段中的第二階段的時間所示。依據一特定實施方式,在聚合酶連鎖反應為等溫環狀擴增法(Loop-Mediated Isothermal Amplification, LAMP)的情況下,如上文表1所示,特定引子可為包含序列辨識號:2至5的第一引子對、或包含序列辨識號:10至13的第二引子對。在另一特定實施方式中,特定引子可為包含序列辨識號:2至7的第一引子對、包含序列辨識號:2至9的第一引子對、包含序列辨識號:10至15的第二引子對、或包含序列辨識號:10至17的第二引子對。該等溫環狀擴增法的反應溫度及時間可如上述的兩階段中的第二階段方式進行的溫度及時間所示。另外,在等溫環狀擴增法中使用的DNA聚合酶可列舉如嗜熱性桿菌屬細菌來源DNA聚合酶而言,有 Bacillus stearothermophilus來源DNA聚合酶(Bst DNA聚合酶)或其經修飾的變異體等。由於等溫環狀擴增法與習知聚合酶連鎖反應主要不同之處在於前者可以在恆溫下進行反應,因而反應溫度不須大幅度升降溫,即能夠具有優異的準確度,並且能以簡易的裝置進行。依據另一特定實施方式,在聚合酶連鎖反應為解旋酶依賴擴增反應(Helicase-dependent Amplification, HDA)的情況下,如上文表1所示,特定引子可為序列辨識號:18及19的第三引子對、序列辨識號:20及21的第四引子對及序列辨識號:22及23的第五引子對。 According to a specific embodiment, when the temperature of the polymerase chain reaction is carried out in two stages, the reaction time of the first stage may be about 1 to 5 minutes, for example, it may be about 2, 3, or 4 minutes; The reaction time can be about 40 to 150 minutes, for example, it can be about 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130 , 135, 140, 145 minutes, but not limited to. However, in another specific embodiment, when the temperature of the polymerase chain reaction is carried out in one stage, the reaction time can be represented by the time of the second stage of the above two stages. According to a specific embodiment, when the polymerase chain reaction is an isothermal loop amplification method (Loop-Mediated Isothermal Amplification, LAMP), as shown in Table 1 above, the specific primers may include sequence identification numbers: 2 to The first primer pair of 5, or the second primer pair comprising SEQ ID NOs: 10 to 13. In another specific embodiment, the specific primers can be the first primer pair comprising SEQ ID NOs: 2 to 7, the first primer pair comprising SEQ ID NOs: 2 to 9, the first primer pair comprising SEQ ID numbers: 10 to 15 A pair of primers, or a second pair of primers comprising SEQ ID NOs: 10 to 17. The reaction temperature and time of the isothermal loop amplification method can be as indicated by the temperature and time of the second stage of the above-mentioned two stages. In addition, the DNA polymerase used in the isothermal loop amplification method includes, for example, DNA polymerase derived from bacteria belonging to the genus Thermophilus, DNA polymerase derived from Bacillus stearothermophilus (Bst DNA polymerase), or modified variants thereof. body etc. The main difference between the isothermal circular amplification method and the conventional polymerase chain reaction is that the former can carry out the reaction at a constant temperature, so the reaction temperature does not need to be greatly increased or decreased, that is, it can have excellent accuracy, and can be easily device is carried out. According to another specific embodiment, when the polymerase chain reaction is a helicase-dependent amplification reaction (Helicase-dependent Amplification, HDA), as shown in Table 1 above, the specific primers can be SEQ ID NOs: 18 and 19 The third primer pair, the fourth primer pair with SEQ ID NOs: 20 and 21, and the fifth primer pair with SEQ ID numbers: 22 and 23.

本文使用的「解旋酶」係可使用所屬技術領域中具有通常知識者已知的解旋酶,其為沿著核酸的磷酸二酯骨架定向移動,使用源自於ATP水解的能量將核酸鏈(例如DNA、RNA或RNA-DNA雜合體)分開的蛋白。所屬技術領域中具有通常知識者能夠選擇適合的具有解旋酶活性的酶用於本揭露的方法。在一實施方式中,解旋酶選自來自於不同家族的解旋酶:可舉出Superfamily I解旋酶(例如dda、pcrA、F質粒tral蛋白解旋酶、uvrD)、Superfamily II解旋酶(例如recQ、NS3解旋酶)、Superfamily III解旋酶(例如AAVrep解旋酶)、來自於DnaB樣Superfamily的解旋酶(例如T7噬菌體解旋酶)或來自於Rho樣Superfamily的解旋酶。As used herein, a "helicase" can be a helicase known to those of ordinary skill in the art, which moves directionally along the phosphodiester backbone of a nucleic acid, using energy derived from ATP hydrolysis to separate nucleic acid strands (eg DNA, RNA or RNA-DNA hybrids) separate proteins. One of ordinary skill in the art can select suitable enzymes with helicase activity for use in the methods of the present disclosure. In one embodiment, the helicase is selected from helicases from different families: Superfamily I helicases (eg dda, pcrA, F plasmid tral protein helicase, uvrD), Superfamily II helicases (e.g. recQ, NS3 helicase), Superfamily III helicases (e.g. AAVrep helicase), helicases from the DnaB-like Superfamily (e.g. T7 phage helicase) or helicases from the Rho-like Superfamily .

由於解旋酶依賴擴增反應與等溫環狀擴增法相似,同樣可以在恆溫下進行反應,因而亦具有能夠在維持優異準確度的同時,能以簡易、方便的裝置進行擴增反應,故有利於在田野調查等環境下應用。Since the helicase-dependent amplification reaction is similar to the isothermal circular amplification method, the reaction can also be carried out at a constant temperature, so the amplification reaction can be carried out with a simple and convenient device while maintaining excellent accuracy. Therefore, it is favorable for application in field investigation and other environments.

在步驟(c)中,判定步驟(b)中經擴增之聚合酶連鎖反應產物中是否具有草莓炭疽菌的DNA基因片段。例如將聚合酶連鎖反應產物以例如凝膠電泳法或螢光等方法分析步驟(b)中經擴增後的聚合酶連鎖反應產物,以確認該聚合酶連鎖反應產物中是否包含特定草莓炭疽菌的DNA基因片段,但本揭露不限於此,例如由於聚合酶連鎖反應產物中可能會產生白色沉澱物,故也可使用例如濁度計、肉眼觀察等的方式進行判斷。In step (c), it is determined whether the amplified polymerase chain reaction product in step (b) has a DNA gene fragment of Anthracis fragariae. For example, the polymerase chain reaction product is analyzed by methods such as gel electrophoresis or fluorescence to analyze the amplified polymerase chain reaction product in step (b) to confirm whether the polymerase chain reaction product contains specific anthracis fragariae However, the present disclosure is not limited to this. For example, since white precipitates may be produced in the polymerase chain reaction product, methods such as turbidimeter and visual observation can also be used to judge.

本揭露之另一態樣提供一種用於草莓炭疽病的檢測裝置。請參照第1至3圖,該檢測裝置100包括:控溫模組110、溫度感測器120以及恆溫槽130,其中該控溫模組110係使用模糊控制程式111以及比例積分微分(PID)控制程式112,其中,控溫模組110與溫度感測器120及恆溫槽130電性連接,且溫度感測器120用以感測恆溫槽130的溫度。溫度感測器120將感測到的溫度變數資料輸出至控溫模組110。當控溫模組110的判斷結果為恆溫槽130的溫度不等於預定溫度時則進行升降溫的調控。舉例而言,為當恆溫槽130的溫度高於預定溫度時,控溫模組110相對應輸出降低熱源的熱源調整訊號;或者恆溫槽130的溫度低於預定溫度時,控溫模組110相對應輸出提高熱源的熱源調整訊號。Another aspect of the present disclosure provides a detection device for strawberry anthracnose. Please refer to FIGS. 1 to 3 , the detection device 100 includes a temperature control module 110 , a temperature sensor 120 and a constant temperature bath 130 , wherein the temperature control module 110 uses a fuzzy control program 111 and a proportional integral derivative (PID) In the control program 112 , the temperature control module 110 is electrically connected with the temperature sensor 120 and the constant temperature bath 130 , and the temperature sensor 120 is used for sensing the temperature of the constant temperature bath 130 . The temperature sensor 120 outputs the sensed temperature variable data to the temperature control module 110 . When the judgment result of the temperature control module 110 is that the temperature of the constant temperature tank 130 is not equal to the predetermined temperature, the temperature increase and decrease regulation is performed. For example, when the temperature of the constant temperature bath 130 is higher than the predetermined temperature, the temperature control module 110 correspondingly outputs a heat source adjustment signal for reducing the heat source; or when the temperature of the constant temperature bath 130 is lower than the predetermined temperature, the temperature control module 110 Corresponds to the heat source adjustment signal that increases the heat source output.

具體而言,控溫模組110中的模糊控制程式111以及PID控制程式112皆同時取得溫度變數資料。在一特定實施例中,溫度變數資料可例如為恆溫槽130的溫度與預定溫度之間大小比較後得到的一誤差訊號、前後兩筆誤差訊號之間大小比較後得到的一誤差變化量訊號等。在一特定實施例中,模糊控制程式111以及PID控制程式112基於自溫度感測器120所得的資料對溫度進行同步但彼此獨立地進行調控。Specifically, both the fuzzy control program 111 and the PID control program 112 in the temperature control module 110 simultaneously obtain the temperature variable data. In a specific embodiment, the temperature variable data can be, for example, an error signal obtained by comparing the magnitude between the temperature of the thermostatic bath 130 and a predetermined temperature, an error change signal obtained by comparing the magnitude of the two error signals before and after, and the like. . In a particular embodiment, the fuzzy control program 111 and the PID control program 112 regulate the temperature synchronously but independently of each other based on data obtained from the temperature sensor 120 .

在一特定實施例中,模糊控制程式111能夠進行將一啟始值調整至一預定控制值的主要調控,而PID控制程式112能夠進行控制啟始值調諧控制,以達到加速控制的作用,而無須從最底值操作,即PID控制程式112係進行微調控的輔助調控,進而使恆溫槽130能夠快速並穩定地維持該預定溫度。In a specific embodiment, the fuzzy control program 111 can perform the main control of adjusting a starting value to a predetermined control value, and the PID control program 112 can perform tuning control of the control starting value to achieve the effect of acceleration control, and It is not necessary to operate from the lowest value, that is, the PID control program 112 performs the auxiliary adjustment of the fine adjustment, so that the constant temperature bath 130 can quickly and stably maintain the predetermined temperature.

在一特定實施例中,模糊控制程式111可將誤差及誤差變化量的精確值等進行模糊化得到模糊控制加權值,並基於預設的模糊矩陣判斷變數位置,進行控制參數的調控,其可將恆溫槽130快速地到達預定溫度。In a specific embodiment, the fuzzy control program 111 can fuzzify the error and the precise value of the error variation to obtain the fuzzy control weighted value, and determine the position of the variable based on the preset fuzzy matrix, and adjust the control parameters. The thermostatic bath 130 is quickly brought to a predetermined temperature.

在另一特定實施例,PID控制程式112可採用重整回饋式(Reset-feedback algorithm)邏輯。如第2圖所示,PID控制程式112可基於誤差、誤差變化量、積分項等參數計算出輸出值,然而因積分項長期累積的誤差值會造成重整停滯,使得程序變數產生過大得超設值(overshoot),因此藉由將輸出值進行回送,再次進行積分回饋(1/T i)1121來修正其積分項,以避免積分項造成飽和而達到避免重整停滯的產生。藉此提高溫度調控的穩定性。 In another particular embodiment, the PID control routine 112 may employ a Reset-feedback algorithm logic. As shown in FIG. 2, the PID control program 112 can calculate the output value based on parameters such as error, error variation, integral term, etc. However, the long-term accumulated error value of the integral term will cause the reformation to stagnate, making the program variables too large to exceed Set the value (overshoot), so by returning the output value, the integral feedback (1/T i ) 1121 is performed again to correct the integral term, so as to avoid the saturation of the integral term and avoid the generation of reformation stagnation. Thereby, the stability of temperature regulation is improved.

在一特定實施例中,控溫模組110可以透過硬體電路搭配韌體或軟體,且控溫模組110中的硬體電路可舉出例如,專用積體電路(application specific IC, ASIC)、場域可程式閘陣列(field programmable gate array, FPGA)或系統單晶片(system-on-a-chip, SOC)等。In a specific embodiment, the temperature control module 110 can be combined with firmware or software through a hardware circuit, and the hardware circuit in the temperature control module 110 can be, for example, an application specific integrated circuit (ASIC) , field programmable gate array (field programmable gate array, FPGA) or system-on-a-chip (system-on-a-chip, SOC) and so on.

在一特定實施例中,恆溫槽130可設有隔熱材,該隔熱材可為片狀或板狀的發泡隔熱材或真空隔熱材,但本揭露不限於此,亦即使用能夠將恆溫槽130內的溫度良好地維持之材料即可。恆溫槽130中設置有一基座131,該基座131中設置至少一個反應槽132,基座131的材料可使用導熱係數高的材料,可例如為銅、鋁等導熱係數高的金屬。反應槽132的數量可根據需要而設置,例如該數量可為4至96個,但本揭露不限於此。再者,反應槽132的尺寸及形狀亦可根據需要而設計,例如可採用與微量離心管符合的形狀與尺寸,可舉出例如0.1 ml、0.2 ml、0.6 ml、1.5 ml的微量離心管,但不限於此。In a specific embodiment, the thermostatic bath 130 may be provided with a heat insulating material, and the heat insulating material may be a sheet-like or plate-like foam heat insulating material or a vacuum heat insulating material, but the present disclosure is not limited to this, that is, using Any material that can maintain the temperature in the thermostatic bath 130 well may be sufficient. A base 131 is set in the constant temperature bath 130, and at least one reaction tank 132 is set in the base 131. The base 131 can be made of materials with high thermal conductivity, such as copper, aluminum and other metals with high thermal conductivity. The number of the reaction tanks 132 can be set as required, for example, the number can be 4 to 96, but the present disclosure is not limited thereto. Furthermore, the size and shape of the reaction tank 132 can also be designed according to needs, for example, the shape and size that conform to the microcentrifuge tube can be adopted, such as 0.1 ml, 0.2 ml, 0.6 ml, 1.5 ml microcentrifuge tubes, But not limited to this.

在一特定實施例中,首先將裝有待測草莓之DNA樣本與一特定引子的微量離心管中置入該反應槽132,對檢測裝置100進行控溫設定,以驅動溫度感測器120對反應槽132進行溫度感測,並利用控溫模組110的模糊控制程式111以及PID控制程式112進行溫控判斷,以將反應槽132的溫度設定成適合該特定引子反應的溫度。接著,在反應槽132內進行聚合酶連鎖反應於一特定時間後,將微量離心管取出,即可以上述分析方式檢測聚合酶連鎖反應產物中是否具有該草莓炭疽菌的DNA基因。In a specific embodiment, a microcentrifuge tube containing the DNA sample of the strawberry to be tested and a specific primer is first placed into the reaction tank 132, and the temperature of the detection device 100 is set to control the temperature to drive the temperature sensor 120 to pair. The temperature of the reaction tank 132 is sensed, and the fuzzy control program 111 and the PID control program 112 of the temperature control module 110 are used to perform temperature control judgment, so as to set the temperature of the reaction tank 132 to a temperature suitable for the reaction of the specific primer. Next, after the polymerase chain reaction is carried out in the reaction tank 132 for a certain period of time, the microcentrifuge tube is taken out, and whether the polymerase chain reaction product has the DNA gene of Anthracis fragariae can be detected by the above analysis method.

根據本揭露之檢測裝置,由於檢測裝置能夠藉由優異的控溫模組而能夠快速且穩定地進行控溫,並且能夠藉由將檢測裝置小型化,因而能夠有利於在例如於田野調查等環境下,仍能夠便於攜帶而快速並準確地進行草莓炭疽病的檢測。According to the detection device of the present disclosure, since the detection device can quickly and stably control the temperature by the excellent temperature control module, and the detection device can be miniaturized, it can be advantageous in environments such as field investigations. It is still easy to carry, fast and accurate detection of strawberry anthracnose.

提供以下實例以輔助熟習此項技術者實施本揭露。即使如此,不應將該等實例視為本揭露之限制,因為本揭露所屬技術領域中具有通常知識者在不背離本揭露之精神或範疇的情況下對本文所討論之實施例進行的修改及變化,而仍屬於本揭露之範圍。The following examples are provided to assist those skilled in the art in implementing the present disclosure. Even so, these examples should not be viewed as limitations of the present disclosure, as modifications to the embodiments discussed herein may be made by persons of ordinary skill in the art to which this disclosure pertains without departing from the spirit or scope of this disclosure and change, and still fall within the scope of this disclosure.

實施例Example

實施例1:引子設計Example 1: Primer Design

首先,發明人基於台灣特有種之草莓炭疽菌中的序列辨識號:1之DNA序列經過比對分析設計而成,所設計之引子可複製出涵蓋草莓炭疽菌之部分的序列,並且如第5圖所示,篩選出5組引子對,其中包含: 第一引子對(下文亦可稱為「L1引子對」):L1-F3(序列辨識號:2)、L1-B3(序列辨識號:3)、L1-FIP(序列辨識號:4)、L1-BIP(序列辨識號:5)、L1-F1c(序列辨識號:6)、L1-B1c(序列辨識號:7)、L1-F2(序列辨識號:8)、L1-B2(序列辨識號:9); 第二引子對(下文亦可稱為「L2引子對」):L2-F3(序列辨識號:10)、L2-B3(序列辨識號:11)、L2-FIP(序列辨識號:12)、L2-BIP(序列辨識號:13)、L2-F1c(序列辨識號:14)、L2-B1c(序列辨識號:15)、L2-F2(序列辨識號:16)、L2-B2(序列辨識號:17); 第三引子對(下文亦可稱為「HP2引子對」):HP2-F(序列辨識號:18)及HP2-R(序列辨識號:19); 第四引子對(下文亦可稱為「HI2引子對」):HI2-F(序列辨識號:20)及HI2-R(序列辨識號:21); 第五引子對(下文亦可稱為「HI3引子對」):HI3-F(序列辨識號:22)及HI2-R(序列辨識號:23)。 First, the inventors designed the DNA sequence based on the sequence identification number: 1 in Taiwan endemic species Strawberry anthracis through alignment analysis, and the designed primers can replicate the sequence covering the strawberry anthracnose part, and as described in Section 5 As shown in the figure, 5 sets of primer pairs were screened, including: The first primer pair (hereinafter also referred to as "L1 primer pair"): L1-F3 (SEQ ID NO: 2), L1-B3 (SEQ ID NO: 3), L1-FIP (SEQ ID NO: 4), L1-BIP (serial identification number: 5), L1-F1c (serial identification number: 6), L1-B1c (serial identification number: 7), L1-F2 (serial identification number: 8), L1-B2 (serial identification number: 8) number: 9); The second primer pair (hereinafter also referred to as "L2 primer pair"): L2-F3 (SEQ ID NO: 10), L2-B3 (SEQ ID NO: 11), L2-FIP (SEQ ID NO: 12), L2-BIP (Serial ID: 13), L2-F1c (Serial ID: 14), L2-B1c (Serial ID: 15), L2-F2 (Serial ID: 16), L2-B2 (Serial ID: 16) number: 17); The third primer pair (hereinafter also referred to as "HP2 primer pair"): HP2-F (SEQ ID NO: 18) and HP2-R (SEQ ID NO: 19); The fourth primer pair (hereinafter also referred to as "HI2 primer pair"): HI2-F (SEQ ID NO: 20) and HI2-R (SEQ ID NO: 21); The fifth primer pair (hereinafter also referred to as "HI3 primer pair"): HI3-F (SEQ ID NO: 22) and HI2-R (SEQ ID NO: 23).

另外,發明人亦基於序列辨識號:1的序列設計出另三組比較例(未示於第5圖),其分別為: 第一比較例(下文亦可稱為「HP1引子對」):HP1-F(序列辨識號:24)及HP1-R(序列辨識號:25); 第二比較例(下文亦可稱為「HP3引子對」):HP3-F(序列辨識號:26)及HP3-R(序列辨識號:27); 第三比較例(下文亦可稱為「HI1引子對」):HI1-F(序列辨識號:28)及HI1-R(序列辨識號:29)。 In addition, the inventor also designed another three sets of comparative examples (not shown in Figure 5) based on the sequence of SEQ ID: 1, which are: The first comparative example (hereinafter also referred to as "HP1 primer pair"): HP1-F (SEQ ID NO: 24) and HP1-R (SEQ ID NO: 25); The second comparative example (hereinafter also referred to as "HP3 primer pair"): HP3-F (SEQ ID NO: 26) and HP3-R (SEQ ID NO: 27); The third comparative example (hereinafter also referred to as "HI1 primer pair"): HI1-F (SEQ ID NO: 28) and HI1-R (SEQ ID NO: 29).

實施例2:聚合酶連鎖反應Example 2: Polymerase Chain Reaction

將所設計的5組引子對中的分成L1引子對及L2引子對以LAMP等溫環狀擴增法進行實驗,另外將HP1引子對、HP2引子對、HP3引子對、HI1引子對、HI2引子對、HI3引子對以兩階段聚合酶連鎖反應及一階段聚合酶連鎖反應進行實驗。Divide the designed primer pairs into L1 primer pair and L2 primer pair by LAMP isothermal circular amplification method. In addition, HP1 primer pair, HP2 primer pair, HP3 primer pair, HI1 primer pair, HI2 primer pair The pair, HI3 primer pairs were tested with two-stage polymerase chain reaction and one-stage polymerase chain reaction.

2.1 LAMP等溫環狀擴增2.1 LAMP isothermal circular amplification

首先,將L1引子對及L2引子對分別如下表2分成P4組、P6組及P8組,在進行核酸擴增之前,進行解離實驗分析,如第6圖所示, L1引子對(A)及L2引子對(B)不論在P4組、P6組及P8組在半解離(Tm)溫度均只有一個Tm值且數值接近,顯示針對聚合酶連鎖反應產物具有專一性。First, the L1 primer pair and the L2 primer pair were divided into P4 group, P6 group and P8 group respectively as shown in Table 2 below. Before nucleic acid amplification, the dissociation experiment was analyzed. As shown in Figure 6, the L1 primer pair (A) and L2 primer pair (B) had only one Tm value at half dissociation (Tm) temperature in P4 group, P6 group and P8 group, and the values were close, indicating the specificity for polymerase chain reaction products.

[表2] 引子名稱 P4 (μM) P6 (μM) P8 (μM) L1/L2-FIP 1.6 1.6 1.6 L1/L2-BIP 1.6 1.6 1.6 L1/L2-F3 0.2 0.2 0.2 L1/L2-B3 0.2 0.2 0.2 L1/L2-F1C - 0.4 0.4 L1/L2-B1C - 0.4 0.4 L1/L2-F2 - - 0.4 L1/L2-B2 - - 0.4 [Table 2] Introductory name P4 (μM) P6 (μM) P8 (μM) L1/L2-FIP 1.6 1.6 1.6 L1/L2-BIP 1.6 1.6 1.6 L1/L2-F3 0.2 0.2 0.2 L1/L2-B3 0.2 0.2 0.2 L1/L2-F1C - 0.4 0.4 L1/L2-B1C - 0.4 0.4 L1/L2-F2 - - 0.4 L1/L2-B2 - - 0.4

接著,在進行LAMP等溫環狀擴增的反應液中,加入含有草莓炭疽菌之草莓DNA樣本(由行政院農業委員會苗栗區農業改良場所提供)並以無菌之去離子水取代DNA樣本作為負控制組,並如上文表2所示的最終引子濃度分別加入L1引子對P4組、P6組、P8組或L2引子對P4組、P6組、P8組,並使用WarmStart LAMP套組(DNA & RNA)(NEB公司製;產品型號:E1700S,其中含有 Bst2.0 WarmStart DNA聚合酶)在65℃下分別反應60分鐘及150分鐘。 Next, in the reaction solution for LAMP isothermal circular amplification, add a strawberry DNA sample containing Strawberry anthracis (provided by the Agricultural Improvement Center of Miaoli District, Agricultural Committee of the Executive Yuan) and replace the DNA sample with sterile deionized water as a negative Control group, and add L1 primer to P4 group, P6 group, P8 group or L2 primer pair P4 group, P6 group, P8 group respectively as the final primer concentration shown in Table 2 above, and use WarmStart LAMP set (DNA & RNA ) (manufactured by NEB Corporation; product model: E1700S, which contains Bst 2.0 WarmStart DNA polymerase) and reacted at 65° C. for 60 minutes and 150 minutes, respectively.

接著取5μL上述LAMP聚合酶連鎖反應之產物,並於其中加入6X的溴酚藍(bromophenol blue)的DNA染料後,將其與5μL 100 bp分子量標準品(ladder marker)分別載入2%洋菜糖膠體,並於1×TAE(0.04 M Tris,0.02 M醋酸鹽(acetate),0.001 M EDTA)緩衝液中進行電泳(100 Volts, 25分鐘)。電泳完成後,將膠體置於含溴化乙錠(ethidium bromide)的水溶液後,於UV燈下照相紀錄。Then take 5μL of the above LAMP polymerase chain reaction product, add 6X bromophenol blue DNA dye to it, and load it and 5μL 100 bp molecular weight standard (ladder marker) into 2% agar. Glycocolloids and electrophoresed (100 Volts, 25 min) in 1×TAE (0.04 M Tris, 0.02 M acetate, 0.001 M EDTA) buffer. After electrophoresis, the colloid was placed in an aqueous solution containing ethidium bromide and photographed under UV light.

如第7圖所示,分別顯示第一引子對P4組、P6組及P8組與第二引子P4組、P6組及P8組進行60分鐘(第7圖(A))及150分鐘(第7圖(B))聚合酶鏈鎖反應的電泳分析結果。自圖式結果可知,自65℃下反應60分鐘的電泳結果可知,在含有草莓炭疽菌之草莓DNA樣本的情況下,第一引子對P4組、P6組及P8組與第二引子對P4組、P6組及P8組可觀察到有梯狀帶現象(ladder-like pattern),而負控制組在上述位置並未出現任何帶狀,顯示第一引子對P4組、P6組及P8組與第二引子P4組、P6組及P8組皆能有效地檢測出草莓炭疽菌的存在,其中以第一引子對及第二引子對的P4組、P6組的梯狀帶現象最為清晰。另外,自65℃下反應150分鐘的電泳結果可知,在含有草莓炭疽菌之草莓DNA樣本與負控制組相比的情況下,第一引子對P4組、P6組及P8組與第二引子對P4組、P6組及P8組雖然稍有模糊(smear)帶狀的現象,但仍能夠辨識出梯狀帶現象,顯示在此條件下仍能有效檢測出草莓炭疽菌的存在。As shown in Fig. 7, the first primer pairs P4, P6 and P8 groups and the second primers P4, P6 and P8 groups were performed for 60 minutes (Fig. 7(A)) and 150 minutes (Fig. 7(A)), respectively. Panel (B)) Electrophoretic analysis results of polymerase chain reaction. As can be seen from the results in the figure, from the electrophoresis results of the reaction at 65°C for 60 minutes, in the case of strawberry DNA samples containing Strawberry anthracis, the first primer pair P4, P6 and P8 groups and the second primer pair P4 group , P6 group and P8 group can be observed to have a ladder-like pattern (ladder-like pattern), while the negative control group did not appear any belt at the above position, showing that the first primer pair P4, P6 and P8 groups and the first The two primers P4, P6 and P8 groups can effectively detect the presence of Strawberry anthracnose, among which the first primer pair and the second primer pair P4 and P6 groups have the clearest ladder band phenomenon. In addition, from the electrophoresis results of the reaction at 65°C for 150 minutes, when the strawberry DNA samples containing strawberry anthracis were compared with the negative control group, the first primer pair P4 group, P6 group and P8 group were compared with the second primer pair. Although the P4, P6 and P8 groups had a slightly smear band, they could still identify the ladder band phenomenon, indicating that the presence of anthracnose can still be effectively detected under this condition.

2.2 二階段控溫的聚合酶鏈鎖反應2.2 Two-stage temperature-controlled polymerase chain reaction

在進行二階段控溫的聚合酶鏈鎖反應的反應液中,加入含有草莓炭疽菌之草莓DNA樣本(由行政院農業委員會苗栗區農業改良場所提供)並以未加入任何DNA樣本作為負控制組,並如下文表3所示的最終引子濃度分別加入HP1引子對、HP2引子對、HP3引子對、HI1引子對、HI2引子對、HI3引子對,並使用IsoAmp II Universal tHDA套組(NEB公司製;產品型號:H0110S)。首先,將上述反應液與套組中的黏合緩衝液II(Annealing buffer II)混合而得到混合液A,將該混合液在95℃下反應2分鐘後置於冰上。接著,將含有黏合緩衝液II、dNTP、MgSO 4、NaCl及IsoAmp酵素的混合液B與經冷卻的混合液A混合,並在65℃下反應90分鐘。 In the reaction solution of the two-stage temperature-controlled polymerase chain reaction, a strawberry DNA sample containing strawberry anthracnose (provided by the Agricultural Improvement Site of Miaoli District, Agricultural Committee of the Executive Yuan) was added, and no DNA sample was added as a negative control group. , and add HP1 primer pair, HP2 primer pair, HP3 primer pair, HI1 primer pair, HI2 primer pair, HI3 primer pair as shown in Table 3 below, respectively, and use IsoAmp II Universal tHDA set (NEB company system) ; Product model: H0110S). First, the above reaction solution was mixed with the Adhesion Buffer II (Annealing buffer II) in the kit to obtain a mixed solution A. The mixed solution was reacted at 95°C for 2 minutes and then placed on ice. Next, mixed solution B containing binding buffer II, dNTP, MgSO 4 , NaCl and IsoAmp enzyme was mixed with cooled mixed solution A, and reacted at 65° C. for 90 minutes.

[表3] 引子名稱 HI1-F/R HI2-F/R HI3-F/R HP1-F/R HP2-F/R HP3-F/R 濃度(nM) 75 75 75 75 75 75 [table 3] Introductory name HI1-F/R HI2-F/R HI3-F/R HP1-F/R HP2-F/R HP3-F/R Concentration (nM) 75 75 75 75 75 75

接著,取5μL上述二階段聚合酶鏈鎖反應之產物,並以與LAMP聚合酶連鎖反應同樣的方式進行電泳,以得到產物的電泳分析結果。Next, 5 μL of the product of the above two-stage polymerase chain reaction was taken and subjected to electrophoresis in the same manner as the LAMP polymerase chain reaction to obtain the electrophoresis analysis result of the product.

如第8圖所示,分別顯示HP1引子對、HP2引子對、HP3引子對、HI1引子對、HI2引子對、HI3引子對的二階段聚合酶鏈鎖反應的電泳分析結果。自圖式結果可知,在含有草莓炭疽菌DNA的草莓樣本中,HP2引子對、HI2引子對、HI3引子對在100至200個bp的位置皆出現明顯的帶狀現象,而負控制組在上述位置皆未出現任何帶狀,顯示HP2引子對、HI2引子對、HI3引子對皆能有效地檢測出草莓炭疽菌的存在。As shown in FIG. 8, the electrophoresis analysis results of the two-stage PCR of the HP1 primer pair, the HP2 primer pair, the HP3 primer pair, the HI1 primer pair, the HI2 primer pair, and the HI3 primer pair are shown, respectively. It can be seen from the graphical results that in the strawberry samples containing the DNA of B. anthracis, the HP2 primer pair, HI2 primer pair, and HI3 primer pair all showed obvious banding at the position of 100 to 200 bp. There is no band at all positions, indicating that the HP2 primer pair, the HI2 primer pair, and the HI3 primer pair can effectively detect the presence of Strawberry anthracnose.

2.3 一階段控溫的聚合酶鏈鎖反應2.3 One-stage temperature-controlled polymerase chain reaction

一階段控溫的聚合酶鏈鎖反應的反應液製備與二階段控溫的聚合酶鏈鎖反應相似,皆使用HP1引子對、HP2引子對、HP3引子對、HI1引子對、HI2引子對、HI3引子對,其差異僅在於省略混合液A及在95℃下加熱的步驟,而直接將反應液與混合液B混合,並在65℃下反應90分鐘。The preparation of the reaction solution for the one-stage temperature-controlled PCR reaction is similar to that of the two-stage temperature-controlled PCR, using the HP1 primer pair, HP2 primer pair, HP3 primer pair, HI1 primer pair, HI2 primer pair, HI3 primer pair The difference between the primer pairs is that the steps of mixing solution A and heating at 95°C are omitted, and the reaction solution and mixture B are directly mixed and reacted at 65°C for 90 minutes.

接著,取5μL上述一階段聚合酶鏈鎖反應之產物,並以與LAMP聚合酶連鎖反應同樣的方式進行電泳,以得到產物的電泳分析結果。Next, 5 μL of the product of the above-mentioned one-stage polymerase chain reaction was taken and subjected to electrophoresis in the same manner as the LAMP polymerase chain reaction to obtain the electrophoresis analysis result of the product.

如第9圖所示,分別顯示HP1引子對、HP2引子對、HP3引子對、HI1引子對、HI2引子對、HI3引子對的一階段聚合酶鏈鎖反應的電泳分析結果。自圖式結果可知,在含有草莓炭疽菌DNA的草莓樣本中,HP2引子對、HI3引子對在100至200個bp的位置皆出現明顯的帶狀現象,而負控制組在上述位置皆未出現任何帶狀,顯示HP2引子對、HI3引子對在僅以一階段控溫的情況下,仍能有效地檢測出草莓炭疽菌的存在。As shown in FIG. 9, the electrophoresis analysis results of the one-stage PCR of the HP1 primer pair, the HP2 primer pair, the HP3 primer pair, the HI1 primer pair, the HI2 primer pair, and the HI3 primer pair are shown, respectively. It can be seen from the results of the figure that in the strawberry samples containing the DNA of B. fragariae, the HP2 primer pair and the HI3 primer pair showed obvious banding at the positions of 100 to 200 bp, while the negative control group did not appear at the above positions. Any band, showing that the HP2 primer pair and the HI3 primer pair can still effectively detect the presence of Strawberry anthracnose under the condition of only one-stage temperature control.

雖然本揭露已以上述實施例進行說明,然其並非用以限定本揭露,在不脫離本發明之精神和範圍內,當所屬領域中具有通常知識者可進行變更與修改,因此本發明之保護範圍當視後附之申請專利範圍所界定者為準。Although the present disclosure has been described with the above-mentioned embodiments, it is not intended to limit the present disclosure. Those with ordinary knowledge in the field can make changes and modifications without departing from the spirit and scope of the present invention. Therefore, the present invention is protected by The scope shall be determined by the scope of the appended patent application.

100:檢測裝置 110:控溫模組 111:模糊控制程式 112:比例積分微分控制程式 1121:積分回饋 120:溫度感測器 130:恆溫槽 131:基座 132:反應槽 100: Detection device 110: Temperature control module 111: Fuzzy Control Program 112: Proportional-Integral-Derivative Control Program 1121: Points feedback 120: temperature sensor 130: Thermostat 131: Pedestal 132: Reaction tank

第1圖顯示根據本揭露之用於檢測草莓炭疽病的檢測裝置之方塊圖。 第2圖顯示根據本揭露之檢測裝置中的比例積分微分控制程式之方塊圖。 第3圖顯示根據本揭露之檢測裝置中的恆溫槽之示意圖。 第4圖顯示根據本揭露之檢測裝置中的反應槽之示意圖。 第5圖顯示根據本揭露之用於檢測草莓炭疽病的引子對之相對位置示意圖。 第6A圖及第6B圖分別顯示根據本揭露之用於檢測草莓炭疽病的第一引子對(L1)增幅產物及第二引子對(L2)增幅產物之解離曲線圖。 第7A圖顯示根據本揭露之用於檢測草莓炭疽病的第一引子對(L1)及第二引子對(L2)進行聚合酶鏈鎖反應60分鐘所獲得之產物進行電泳分析的結果。 第7B圖顯示根據本揭露之用於檢測草莓炭疽病的第一引子對(L1)及第二引子對(L2)進行聚合酶鏈鎖反應150分鐘所獲得之產物進行電泳分析的結果。 第8圖顯示根據本揭露之用於檢測草莓炭疽病的第三引子對(HP2)、第四引子對(HI2)、第五引子對(HI3)及各比較例進行二階段控溫的聚合酶鏈鎖反應所獲得之產物進行電泳分析的結果。 第9圖顯示根據本揭露之用於檢測草莓炭疽病的第三引子對(HP2)、第四引子對(HI2)、第五引子對(HI3)及各比較例進行一階段控溫的聚合酶鏈鎖反應所獲得之產物進行電泳分析的結果。 FIG. 1 shows a block diagram of a detection device for detecting strawberry anthracnose according to the present disclosure. FIG. 2 shows a block diagram of a proportional-integral-derivative control program in the detection device according to the present disclosure. FIG. 3 shows a schematic diagram of a constant temperature bath in the detection device according to the present disclosure. FIG. 4 shows a schematic diagram of the reaction tank in the detection device according to the present disclosure. FIG. 5 shows a schematic diagram of the relative positions of primer pairs for detecting strawberry anthracnose according to the present disclosure. 6A and 6B respectively show dissociation curves of the amplified product of the first primer pair (L1) and the amplified product of the second primer pair (L2) for detecting strawberry anthracnose according to the present disclosure. Figure 7A shows the results of electrophoresis analysis of the products obtained by carrying out the polymerase chain reaction of the first primer pair (L1) and the second primer pair (L2) for detecting strawberry anthracnose for 60 minutes according to the present disclosure. FIG. 7B shows the result of electrophoresis analysis of the products obtained by the PCR for 150 minutes for the first primer pair (L1) and the second primer pair (L2) for detecting strawberry anthracnose according to the present disclosure. Fig. 8 shows the third primer pair (HP2), the fourth primer pair (HI2), the fifth primer pair (HI3) for detecting strawberry anthracnose according to the present disclosure and the polymerases of each comparative example for two-stage temperature control The result of electrophoresis analysis of the products obtained by the chain reaction. Figure 9 shows the third primer pair (HP2), the fourth primer pair (HI2), the fifth primer pair (HI3) for detecting strawberry anthracnose according to the present disclosure, and the polymerases of each comparative example for one-stage temperature control The result of electrophoresis analysis of the products obtained by the chain reaction.

<110> 財團法人工業技術研究院、行政院農業委員會苗栗區農業改良場 <110> Industrial Technology Research Institute, Executive Yuan Agriculture Committee Miaoli District Agricultural Improvement Field

<120> 用於檢測草莓炭疽病的方法、引子對、以及檢測裝置 <120> Method, primer pair, and detection device for detecting strawberry anthracnose

<160> 29 <160> 29

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

<210> 1 <210> 1

<211> 1161 <211> 1161

<212> DNA <212> DNA

<213> 草莓炭疽菌(Colletotrichum siamense) <213> Colletotrichum siamense

<400> 1 <400> 1

<400> 1

Figure 109146642-A0305-02-0024-1
Figure 109146642-A0305-02-0025-2
<400> 1
Figure 109146642-A0305-02-0024-1
Figure 109146642-A0305-02-0025-2

<210> 2 <210> 2

<211> 19 <211> 19

<212> DNA <212> DNA

<213> 人工序列(Artificial sequence) <213> Artificial sequence

<220> <220>

<223> 用於LAMP方法的引子L1-F3 <223> Primers L1-F3 for LAMP method

<400> 2

Figure 109146642-A0305-02-0025-3
<400> 2
Figure 109146642-A0305-02-0025-3

<210> 3 <210> 3

<211> 20 <211> 20

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> 用於LAMP方法的引子L1-B3 <223> Primers L1-B3 for LAMP method

<400> 3

Figure 109146642-A0305-02-0026-4
<400> 3
Figure 109146642-A0305-02-0026-4

<210> 4 <210> 4

<211> 46 <211> 46

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> 用於LAMP方法的引子L1-FIP <223> Primer L1-FIP for LAMP method

<400> 4

Figure 109146642-A0305-02-0026-5
<400> 4
Figure 109146642-A0305-02-0026-5

<210> 5 <210> 5

<211> 48 <211> 48

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> 用於LAMP方法的引子L1-BIP <223> Primer L1-BIP for LAMP method

<400> 5

Figure 109146642-A0305-02-0026-6
<400> 5
Figure 109146642-A0305-02-0026-6

<210> 6 <210> 6

<211> 20 <211> 20

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> 用於LAMP方法的引子L1-F1c <223> Primer L1-F1c for LAMP method

<400> 6

Figure 109146642-A0305-02-0027-7
<400> 6
Figure 109146642-A0305-02-0027-7

<210> 7 <210> 7

<211> 22 <211> 22

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> 用於LAMP方法的引子L1-B1c <223> Primer L1-B1c for LAMP method

<400> 7

Figure 109146642-A0305-02-0027-8
<400> 7
Figure 109146642-A0305-02-0027-8

<210> 8 <210> 8

<211> 20 <211> 20

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> 用於LAMP方法的引子L1-F2 <223> Primers L1-F2 for LAMP method

<400> 8

Figure 109146642-A0305-02-0027-9
<400> 8
Figure 109146642-A0305-02-0027-9

<210> 9 <210> 9

<211> 20 <211> 20

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> 用於LAMP方法的引子L1-B2 <223> Primers L1-B2 for LAMP method

<400> 9

Figure 109146642-A0305-02-0028-10
<400> 9
Figure 109146642-A0305-02-0028-10

<210> 10 <210> 10

<211> 18 <211> 18

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> 用於LAMP方法的引子L2-F3 <223> Primers L2-F3 for LAMP method

<400> 10

Figure 109146642-A0305-02-0028-11
<400> 10
Figure 109146642-A0305-02-0028-11

<210> 11 <210> 11

<211> 20 <211> 20

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> 用於LAMP方法的引子L2-B3 <223> Primers L2-B3 for LAMP method

<400> 11

Figure 109146642-A0305-02-0028-12
<400> 11
Figure 109146642-A0305-02-0028-12

<210> 12 <210> 12

<211> 47 <211> 47

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> 用於LAMP方法的引子L2-FIP <223> Primer L2-FIP for LAMP method

<400> 12

Figure 109146642-A0305-02-0029-13
<400> 12
Figure 109146642-A0305-02-0029-13

<210> 13 <210> 13

<211> 47 <211> 47

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> 用於LAMP方法的引子L2-BIP <223> Primer L2-BIP for LAMP method

<400> 13

Figure 109146642-A0305-02-0029-14
<400> 13
Figure 109146642-A0305-02-0029-14

<210> 14 <210> 14

<211> 22 <211> 22

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> 用於LAMP方法的引子L2-F1c <223> Primer L2-F1c for LAMP method

<400> 14

Figure 109146642-A0305-02-0029-15
<400> 14
Figure 109146642-A0305-02-0029-15

<210> 15 <210> 15

<211> 21 <211> 21

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> 用於LAMP方法的引子L2-B1c <223> Primer L2-B1c for LAMP method

<400> 15

Figure 109146642-A0305-02-0030-16
<400> 15
Figure 109146642-A0305-02-0030-16

<210> 16 <210> 16

<211> 19 <211> 19

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> 用於LAMP方法的引子L2-F2 <223> Primer L2-F2 for LAMP method

<400> 16

Figure 109146642-A0305-02-0030-17
<400> 16
Figure 109146642-A0305-02-0030-17

<210> 17 <210> 17

<211> 20 <211> 20

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> 用於LAMP方法的引子L2-B2 <223> Primer L2-B2 for LAMP method

<400> 17

Figure 109146642-A0305-02-0030-18
<400> 17
Figure 109146642-A0305-02-0030-18

<210> 18 <210> 18

<211> 27 <211> 27

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> HP2-F正向引子 <223> HP2-F forward primer

<400> 18

Figure 109146642-A0305-02-0031-19
<400> 18
Figure 109146642-A0305-02-0031-19

<210> 19 <210> 19

<211> 27 <211> 27

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> HP2-R反向引子 <223> HP2-R reverse primer

<400> 19

Figure 109146642-A0305-02-0031-20
<400> 19
Figure 109146642-A0305-02-0031-20

<210> 20 <210> 20

<211> 23 <211> 23

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> HI2-F正向引子 <223> HI2-F forward primer

<400> 20

Figure 109146642-A0305-02-0031-21
<400> 20
Figure 109146642-A0305-02-0031-21

<210> 21 <210> 21

<211> 27 <211> 27

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> HI2-R反向引子 <223> HI2-R reverse primer

<400> 21

Figure 109146642-A0305-02-0032-22
<400> 21
Figure 109146642-A0305-02-0032-22

<210> 22 <210> 22

<211> 22 <211> 22

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> HI3-F正向引子 <223> HI3-F forward primer

<400> 22

Figure 109146642-A0305-02-0032-23
<400> 22
Figure 109146642-A0305-02-0032-23

<210> 23 <210> 23

<211> 23 <211> 23

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> HI3-R反向引子 <223> HI3-R reverse primer

<400> 23

Figure 109146642-A0305-02-0032-24
<400> 23
Figure 109146642-A0305-02-0032-24

<210> 24 <210> 24

<211> 27 <211> 27

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> HP1-F正向引子 <223> HP1-F forward primer

<400> 24

Figure 109146642-A0305-02-0033-25
<400> 24
Figure 109146642-A0305-02-0033-25

<210> 25 <210> 25

<211> 27 <211> 27

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> HP1-R反向引子 <223> HP1-R reverse primer

<400> 25

Figure 109146642-A0305-02-0033-26
<400> 25
Figure 109146642-A0305-02-0033-26

<210> 26 <210> 26

<211> 27 <211> 27

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> HP3-F正向引子 <223> HP3-F forward primer

<400> 26

Figure 109146642-A0305-02-0033-27
<400> 26
Figure 109146642-A0305-02-0033-27

<210> 27 <210> 27

<211> 27 <211> 27

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> HP3-R反向引子 <223> HP3-R reverse primer

<400> 27

Figure 109146642-A0305-02-0034-28
<400> 27
Figure 109146642-A0305-02-0034-28

<210> 28 <210> 28

<211> 23 <211> 23

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> HI1-F正向引子 <223> HI1-F forward primer

<400> 28

Figure 109146642-A0305-02-0034-29
<400> 28
Figure 109146642-A0305-02-0034-29

<210> 29 <210> 29

<211> 24 <211> 24

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequences

<220> <220>

<223> HI1-R反向引子 <223> HI1-R reverse primer

<400> 29

Figure 109146642-A0305-02-0034-30
<400> 29
Figure 109146642-A0305-02-0034-30

Claims (27)

一種用於檢測草莓炭疽病的方法,其包括以下步驟:(a)獲得一待測草莓之DNA樣本;(b)將該待測草莓之DNA樣本作為模版以一特定引子對進行聚合酶連鎖反應,以獲得一聚合酶連鎖反應產物;以及(c)檢測該聚合酶連鎖反應產物中是否具有一草莓炭疽菌的DNA基因,該草莓炭疽菌的DNA基因包含序列辨識號:1之草莓炭疽菌(Colletotrichum siamense);其中該特定引子對為包含序列辨識號:2至序列辨識號:5的第一引子對、包含序列辨識號:10至序列辨識號:13的第二引子對、序列辨識號:18及19的第三引子對、序列辨識號:20及21的第四引子對、或序列辨識號:22及23的第五引子對;其中該聚合酶連鎖反應為環介導等溫擴增反應(LAMP)或解旋酶依賴擴增反應(HDA)。 A method for detecting strawberry anthracnose, comprising the following steps: (a) obtaining a DNA sample of the strawberry to be tested; (b) using the DNA sample of the strawberry to be tested as a template to carry out a polymerase chain reaction with a specific primer pair , to obtain a polymerase chain reaction product; and (c) detect whether the polymerase chain reaction product has a DNA gene of Strawberry anthracnose, the strawberry anthracnose DNA gene comprising SEQ ID NO: 1: Strawberry anthracnose ( Colletotrichum siamense ); wherein the specific primer pair is the first primer pair comprising SEQ ID NO: 2 to SEQ ID NO: 5, the second primer pair comprising SEQ ID NO: 10 to SEQ ID NO: 13, SEQ ID NO: The third primer pair of 18 and 19, the fourth primer pair of SEQ ID NOs: 20 and 21, or the fifth primer pair of SEQ ID numbers: 22 and 23; wherein the polymerase chain reaction is loop-mediated isothermal amplification reaction (LAMP) or helicase-dependent amplification reaction (HDA). 如請求項1所述之方法,其中該聚合酶連鎖反應的反應溫度為60~75℃。 The method according to claim 1, wherein the reaction temperature of the polymerase chain reaction is 60-75°C. 如請求項1所述之方法,其中該聚合酶連鎖反應的反應時間為40至150分鐘。 The method of claim 1, wherein the reaction time of the polymerase chain reaction is 40 to 150 minutes. 如請求項1所述之方法,其中該第一引子對更進一步包括序列辨識號:6至序列辨識號:7。 The method of claim 1, wherein the first primer pair further comprises SEQ ID NO: 6 to SEQ ID NO: 7. 如請求項4所述之方法,其中該第一引子對更進一步包括序列辨識號:8至序列辨識號:9。 The method of claim 4, wherein the first primer pair further comprises SEQ ID NO: 8 to SEQ ID NO: 9. 如請求項1所述之方法,其中該第二引子對更進一步包括序列辨識號:14至序列辨識號:15。 The method of claim 1, wherein the second primer pair further comprises SEQ ID NO: 14 to SEQ ID NO: 15. 如請求項6所述之方法,其中該第二引子對更進一步包括序列辨識號:16至序列辨識號:17。 The method of claim 6, wherein the second primer pair further comprises SEQ ID NO: 16 to SEQ ID NO: 17. 如請求項1、4至7項中任一項所述之方法,其中該聚合酶連鎖反應為環介導等溫擴增反應(LAMP)。 The method of any one of claims 1, 4 to 7, wherein the polymerase chain reaction is a loop-mediated isothermal amplification reaction (LAMP). 如請求項8所述之方法,其中該聚合酶連鎖反應產物在核酸電泳下呈梯狀帶現象。 The method of claim 8, wherein the polymerase chain reaction product exhibits a ladder-like band phenomenon under nucleic acid electrophoresis. 如請求項1所述之方法,其中該聚合酶連鎖反應係為解旋酶依賴擴增反應(HDA)。 The method of claim 1, wherein the polymerase chain reaction is a helicase-dependent amplification reaction (HDA). 如請求項10所述之方法,其中該聚合酶連鎖反應產物的片段為200個以下鹼基對(bp)。 The method of claim 10, wherein the fragment of the polymerase chain reaction product is less than 200 base pairs (bp). 如請求項10所述之方法,其中該特定引子對為第三引子對或第五引子對。 The method of claim 10, wherein the specific primer pair is the third primer pair or the fifth primer pair. 一種用於檢測草莓炭疽病的引子對,其包括序列辨識號:2至序列辨識號:5的引子對。 A primer pair for detecting strawberry anthracnose comprises the primer pair of SEQ ID NO: 2 to SEQ ID NO: 5. 如請求項13所述之引子對,其更進一步包括序列辨識號:6至序列辨識號:7。 The primer pair of claim 13, further comprising sequence identification number: 6 to sequence identification number: 7. 如請求項13或14所述之引子對,其更進一步包括序列辨識號:8至序列辨識號:9。 The primer pair of claim 13 or 14, further comprising a sequence identification number: 8 to a sequence identification number: 9. 一種用於檢測草莓炭疽病的引子對,其包括序列辨識號:10至序列辨識號:13的引子對。 A primer pair for detecting strawberry anthracnose comprises a primer pair of SEQ ID NO: 10 to SEQ ID NO: 13. 如請求項16所述之引子對,其更進一步包括序列辨識號:14至序列辨識號:15。 The primer pair of claim 16, further comprising SEQ ID NO: 14 to SEQ ID NO: 15. 如請求項16或17所述之引子對,其更進一步包括序列辨識號:16至序列辨識號:17。 The primer pair of claim 16 or 17, further comprising sequence identification number: 16 to sequence identification number: 17. 一種用於檢測草莓炭疽病的引子對,其為序列辨識號:18至序列辨識號:19的引子對。 A primer pair for detecting strawberry anthracnose is the primer pair of SEQ ID NO: 18 to SEQ ID NO: 19. 一種用於檢測草莓炭疽病的引子對,其為序列辨識號:20至序列辨識號:21的引子對。 A primer pair for detecting strawberry anthracnose is a primer pair of SEQ ID NO: 20 to SEQ ID NO: 21. 一種用於檢測草莓炭疽病的引子對,其為序列辨識號:22至序列辨識號:23的引子對。 A primer pair for detecting strawberry anthracnose is the primer pair of SEQ ID NO: 22 to SEQ ID NO: 23. 一種用於檢測草莓炭疽病的檢測裝置,其包括:一控溫模組,其使用一模糊控制程式及一比例積分微分(PID)控制程式進行調控;以及一恆溫槽,其與該控溫模組電性連接,該恆溫槽中設置有至少一個反應槽,其中將一待測草莓之DNA樣本與一特定引子對置入該反應槽,以在一特定溫度下進行聚合酶連鎖反應,其中該聚合酶連鎖反應為環介導等溫擴增反應(LAMP)或解旋酶依賴擴增反應(HDA);其中該特定引子對係為如請求項13至21中任一項所述之引 子對。 A detection device for detecting strawberry anthracnose, comprising: a temperature control module, which uses a fuzzy control program and a proportional-integral-derivative (PID) control program for regulation; and a constant temperature tank, which is connected with the temperature control module The group is electrically connected, and at least one reaction tank is arranged in the constant temperature tank, wherein a DNA sample of strawberry to be tested and a specific primer pair are placed in the reaction tank to carry out a polymerase chain reaction at a specific temperature, wherein the The polymerase chain reaction is loop-mediated isothermal amplification (LAMP) or helicase-dependent amplification (HDA); wherein the specific primer pair is a primer as described in any one of claims 13 to 21 child pair. 如請求項22所述之檢測裝置,其中該模糊控制程式與該PID控制程式同步且獨立地進行溫度調控。 The detection device of claim 22, wherein the fuzzy control program and the PID control program synchronize and independently perform temperature regulation. 如請求項22所述之檢測裝置,其中該PID控制程式採用重整回饋式(Reset-feedback algorithm)邏輯。 The detection device of claim 22, wherein the PID control program uses Reset-feedback algorithm logic. 如請求項22所述之檢測裝置,其中該恆溫槽設有隔熱材。 The detection device according to claim 22, wherein the thermostatic bath is provided with a heat insulating material. 如請求項22所述之檢測裝置,其中該恆溫槽的數量為4至96個。 The detection device of claim 22, wherein the number of the constant temperature baths is 4 to 96. 如請求項22所述之檢測裝置,其中該特定溫度為60~75℃。 The detection device according to claim 22, wherein the specific temperature is 60-75°C.
TW109146642A 2020-12-29 2020-12-29 Primer pair, method and detecting device for detecting colletotrichum siamense TWI765501B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
TW109146642A TWI765501B (en) 2020-12-29 2020-12-29 Primer pair, method and detecting device for detecting colletotrichum siamense

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
TW109146642A TWI765501B (en) 2020-12-29 2020-12-29 Primer pair, method and detecting device for detecting colletotrichum siamense

Publications (2)

Publication Number Publication Date
TWI765501B true TWI765501B (en) 2022-05-21
TW202225413A TW202225413A (en) 2022-07-01

Family

ID=82594246

Family Applications (1)

Application Number Title Priority Date Filing Date
TW109146642A TWI765501B (en) 2020-12-29 2020-12-29 Primer pair, method and detecting device for detecting colletotrichum siamense

Country Status (1)

Country Link
TW (1) TWI765501B (en)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108085410A (en) * 2018-01-10 2018-05-29 浙江农林大学 Seedling stage Strawberry anthracnose latent infection and its fast detection method of medication

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108085410A (en) * 2018-01-10 2018-05-29 浙江农林大学 Seedling stage Strawberry anthracnose latent infection and its fast detection method of medication

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
期刊 Gang Qin, Yanni Ma, Xiangzhou Zhang, Ming Zhang.Design of Fuzzy Adaptive PID Temperature Controller Based on FPGA. Indonesian Journal of Electrical Engineering and Computer Science. Vol. 11. TELKOMNIKA. 2013. 6008-6016.; *
期刊 Pei‑Che Chung, Hung‑Yi Wu, Yen‑Wen Wang, Hiran A. Ariyawansa, Hsien‑Pin Hu, Ting‑Hsuan Hung, Shean‑Shong Tzean, Chia‑Lin Chung.Diversity and pathogenicity of Colletotrichum species causing strawberry anthracnose in Taiwan and description of a new species, Colletotrichum miaoliense sp. nov. Scientific Reports. Vol. 10. Nature. 2020 Sep 4. 14664.; *
期刊 Ziyan Chen, Xiaoli Liu, Qunlin Zhang, Benjin Li.RAPID AND VISUAL DETECTION OF COLLETOTRICHUM GLOEOSPORIOIDES ON ANOECTOCHILUS USING LOOP-MEDIATED ISOTHERMAL AMPLIFICATION ASSAY. International Journal of Phytopathology. Vol. 5. EScience Press. 2017 May 19. 99-106.; *
期刊 鐘珮哲、吳竑毅、曹嘉惠.偵測潛伏感染期草莓炭疽病病原菌技術之研發 行政院農業委員會苗栗區農業改良場研究彙報. 第9期. 行政院農業委員會苗栗區農業改良場. 202006. 46-57. *

Also Published As

Publication number Publication date
TW202225413A (en) 2022-07-01

Similar Documents

Publication Publication Date Title
ES2399054T3 (en) Methods to simplify microbial nucleic acids by chemical modification of cytosines
CN105734164B (en) A kind of multiple PCR reagent kit of detection bacterium meningitis pathogen
US6867021B2 (en) Multiplex RT-PCR/PCR for simultaneous detection of bovine coronavirus, bovine rotavirus, Cryptosporidium parvum, and Escherichia coli
CN110669870B (en) Real-time fluorescent quantitative RT-PCR detection primer, probe and detection kit for Pariemam serogroup virus serotypes
CN102643910B (en) Application of asymmetric multicolor fluorescence hairpin probe chain reaction in pathogenic bacterium detection
KR101712451B1 (en) Method for detecting food borne pathogens using digital PCR
CN115747353A (en) Primer group, reagent, kit and detection method for detecting Listeria monocytogenes
Short et al. Quantitative analysis of nifH genes and transcripts from aquatic environments
CN109777861A (en) The loop-mediated isothermal amplification method of mispairing tolerance and application
TWI765501B (en) Primer pair, method and detecting device for detecting colletotrichum siamense
WO2022068785A1 (en) Nucleic acid detection method for identifying bacillus cereus and bacillus thuringiensis
CN110863061A (en) Specific LAMP primer, kit and method for detecting staphylococcus aureus
CN110878373A (en) Recombinase polymerase amplification detection kit for phytophthora infestans and application thereof
CN116377092A (en) Single-tube nested qPCR reagent for detecting Brucella
CN114958835A (en) Combination product and kit for detecting bacterial rice blight bacteria
CN112280878B (en) Specific target spot, primer, detection method and application for detecting vibrio parahaemolyticus
WO2008122097A1 (en) Synthesis of standards for detection and quantification of nucleic acids and kit
CN115029470A (en) Combination product and kit for detecting rice bacterial blight
CN108060244A (en) A kind of nucleotide sequence and application for mycobacterium tuberculosis complex detection
CN110804674B (en) Primer probe composition and kit for detecting soybean root rot based on recombinase polymerase amplification method and application of primer probe composition and kit
CN104774960A (en) Method for applying dual high-resolution melting curve technology to detect Bartonella
CN111254187A (en) Multiple PCR detection method for Klebsiella pneumoniae, salmonella and bordetella pneumoniae of rabbit
CN112029829A (en) Nucleic acid isothermal amplification method based on hairpin structure and application of kit thereof
Nurjayadi et al. grxB gene as a potential target for the development of Cronobacter sakazakii detection method in infant formula milk using real-time PCR
CN108642190A (en) Forensic medicine composite detection kit based on 14 autosome SNP genetic markers