草莓白粉病菌LAMP可视化快速检测方法的建立及初步应用

doi:10.7668/hbnxb.20193522

摘要/Abstract

摘要：

由羽衣草单囊壳菌引起的草莓白粉病是保护地草莓发病面积较大、发病频率较高的草莓病害之一,从苗期到结果期都能发生危害。建立快速、简便、高效的草莓白粉病病原菌检测技术对于该病的有效诊断和防控尤为重要。以羽衣草单囊壳菌ITS基因保守序列为靶基因,设计了1组包括F3/B3、FIP/BIP和LF/LB的环介导等温扩增(LAMP)特异性引物,通过实时荧光曲线和荧光显色双重判定方法,对体系中影响检测效率的主要因素dNTPs、MgSO₄、BstDNA聚合酶、甜菜碱及温度进行优化,并对优化后的方法进行特异性、灵敏性检测及田间样本检测效果评估。结果表明,建立了草莓白粉病菌LAMP检测体系后,其优化的dNTPs终浓度为1.6 mmol/L,MgSO₄终浓度为8 mmol/L,BstDNA聚合酶终浓度为320 U/moL,甜菜碱终浓度为1.2 mmol/L,反应最佳温度为65 ℃。该方法能够特异性地检测出草莓白粉病病原菌,检测灵敏度达到3.2×10^-4 ng/μL,最低检测浓度可在1 h内完成,效率是普通PCR的100倍。通过LAMP荧光显色法进行草莓白粉病田间样品检测,可有效地检测草莓白粉病发病症状不明显的初侵染样本。该方法具有检测时间短、肉眼直接观察结果、对操作人员要求低、检测成本低等优点,对于草莓白粉病早期诊断、监测,提早预防、确定最佳防治时间具有重要的指导意义。

关键词: 草莓白粉病, 羽衣草单囊壳菌, 环介导等温扩增检测技术(LAMP), 检测

Abstract:

Strawberry powdery mildew caused by Sphaerotheca aphanis is one of the strawberry diseases with large incidence area and high incidence frequency in the protected cultivation areas, which usually occurs from seedling stage to fruiting stage. Therefore, the establishment of a rapid and efficient detection method of S. aphanis is quite important for the diagnosis and control of strawberry powdery mildew.A set of LAMP amplification primers including F3/B3、FIP/BIP and LF/LB were designed for the conserved sequence of ITS gene of Sphaerotheca aphanis as the target gene. Through the double judgment method of Real-time fluorescence curve and fluorescence color change, the main factors in the LAMP reaction system including the final concentrations of dNTPs, MgSO₄, BstDNA polymerase and betaine and amplification temperature were selected and optimized to increased etections ensitivity and specificity. Finally, the effects of field samples detection by optimized LAMP were evaluated.The results showed that the LAMP detection system of strawberry powdery mildew was established successfully. The optimized reaction conditions included dNTPs 1.6 mmol/L, MgSO₄ 8 mmol/L, BstDNA polymerase 320 U/moL and betaine 1.2 mmol/L and the amplification temperature was 65 ℃. Under such conditions, the minimum detection sensitivity was 3.2×10^-4 ng/μL within 60 min, and the efficiency of which was 100 times higher than the result of PCR amplification.The visual LAMP method could effectively detect the samples with no obvious symptoms infected by S. aphanis in the early stage. This method has the advantages of short detection time, direct observation with eyes, low requirements for operators and low detection cost. It has important guiding significance for the early diagnosis, monitoring, early prevention and determination of the best control time of strawberry powdery mildew.

Key words: Strawberry powdery mildew, Sphaerotheca aphanis, Loop-mediateddisothermal amplification (LAMP), Detection

贲海燕, 郝永娟, 霍建飞, 姚玉荣, 高苇, 王万立. 草莓白粉病菌LAMP可视化快速检测方法的建立及初步应用[J]. 华北农学报, 2022, 37(6): 182-190. doi: 10.7668/hbnxb.20193522.

BEN Haiyan, HAO Yongjuan, HUO Jianfei, YAO Yurong, GAO Wei, WANG Wanli. Establishment and Application of Visual LAMP Method for Rapid Detection of Sphaerotheca aphanis Causing Strawberry Powdery Mildew[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(6): 182-190. doi: 10.7668/hbnxb.20193522.

http://www.hbnxb.net/CN/Y2022/V37/I6/182

图/表 11

表1 用于草莓白粉病菌LAMP测试设计的引物组信息

Tab.1 Designed primer set for LAMP detection of strawberry powdery mildew

引物名称 Prime name	引物序列(5'—3') Prime sequence(5'—3')	引物组碱基长度/bp Prime set length of base
F3-1	CGGTATTCCGAGGGGCAT	18
B3-1	CCTGATCCGAGGTCATCCAA	20
FIP-1	CTGTTTAGGGGACGCCGAGCTGTTCGAGCGTCAGAACATC	40
BIP-1	GGCGGTACCGTTGTGCTCTCTAGGTGGGTTTTGGCAGGT	38
LF-1	ACCAAGCCAGGCTTGAGAG	19
LB-1	GTAGTCACGTATCTCGCGACAGAG	24

图1 LAMP检测体系中dNTPs终浓度的优化结果 A.实时荧光曲线结果;B.反应液荧光显色结果(编号1—6分别为dNTPs终浓度0.8,1.0,1.2,1.4,1.6,1.8 mmol/L;N为阴性对照)。

Fig.1 Optimized results of LAMP detection system for final concentration of dNTPs A.The curve results of Real-time fluorescence; B.Fluorescent color change results of reaction solution(Numbers 1—6 were the final dNTP concentrations of 0.8,1.0,1.2,1.4,1.6,1.8 mmol/L,respectively;N was the negative control).

图2 LAMP检测体系中Mg²⁺终浓度的优化结果 A.实时荧光曲线结果;B.反应液荧光显色结果(编号1~5分别为Mg²⁺终浓度4,6,8,10,12 mmol/L;N为阴性对照)。

Fig.2 Optimized results of LAMP detection system for final concentration of Mg²⁺ A.The curve results of Real-time fluorescence; B.Fluorescent color change results of reaction solution(Numbers 1—5 were the final Mg²⁺ concentrations of 4,6,8,10,12 mmol/L,respectively;N was the negative control).

图3 LAMP检测体系中甜菜碱终浓度的优化结果 A.实时荧光曲线结果;B.反应液荧光显色结果(编号1~7分别为甜菜碱终浓度0,0.2,0.4,0.6,0.8,1.0,1.2 mmol/L;N为阴性对照)。

Fig.3 Optimized results of LAMP detection system for final concentration of betaine A.The curve results of Real-time fluorescence; B.Fluorescent color change results of reaction solution(Numbers 1—7 were the final betaine concentrations of 0,0.2,0.4,0.6,0.8,1.0,1.2 mmol/L,respectively,N was the negative control).

图4 LAMP检测体系中BstDNA聚合酶终浓度的优化结果 A.实时荧光曲线结果;B.反应液荧光显色结果(编号1~5分别为BstDNA聚合酶终浓度40,80,160,320,340 U/mL;N为阴性对照)。

Fig.4 Optimized results of LAMP detection system for final concentration of BstDNA polymerase A.The curve results of Real-time fluorescence; B.Fluorescent color change results of reaction solution(Numbers 1—5 were the final BstDNA polymerase concentrations of 40,80,160,320,340 U/mL,respectively;N was the negative control).

图5 LAMP检测体系中温度的优化结果 A.实时荧光曲线.;B.反应液荧光显色结果(编号1~3分别为温度63,65,67 ℃;N为阴性对照)。

Fig.5 Optimized results of LAMP detection system for final concentration of temperature A.The curve results of Real-time fluorescence; B.Fluorescent color change results of reaction solution (Numbers 1—3 were temperatures of 63,65,67 ℃,respectively;N was the negative control).

图6 草莓白粉病菌LAMP引物特异性检测结果 A.实时荧光曲线结果;B.反应液荧光显色结果(编号1为草莓白粉病菌;2~7依次为对照菌株辣椒白粉病菌、黄瓜白粉病菌、草莓灰霉病菌、草莓根腐病菌、草莓炭疽病菌、草莓蛇眼病菌;N为阴性对照)。

Fig.6 Detection results of lamp primer specificity of strawberry powdery mildew A.The curve results of Real-time fluorescence; B.Fluorescent color change results of reaction solution (Number 1 was strawberry powdery mildew(Sphaerotheca aphanis),2—7 were the control strains,followed by powdery mildew of pepper(Oidiopsis taurica),cucumber powdery mildew(Sphaerotheca cucurbitae),strawberry gray mold(Botrytis cinerea),strawberry root rot(Fusarium sp.),strawberry anthracnose(Colletotrichum gloeosporioides),white leaf spot of strawberry(Mycosphaerella fragariae),respectively;N was the negative control).

图7 草莓白粉病菌LAMP扩增灵敏度的检测结果 A.实时荧光曲线结果;B.反应液荧光显色结果(编号1~7草莓病原菌粗体DNA浓度依次为3.2,3.2×10^-1,3.2×10^-2,3.2×10^-3,3.2×10^-4,3.2×10^-5,3.2×10^-6ng/μL;N为阴性对照)。

Fig.7 Detection results of sensitivity of strawberry powdery mildew by using lamp A.The curve results of Real-time fluorescence; B.Fluorescent color change results of reaction solution(The bold DNA concentrations of strawberry pathogens numbered 1—7 were 3.2,3.2×10^-1,3.2×10^-2,3.2×10^-3,3.2×10^-4,3.2×10^-5,3.2×10^-6ng/μL,respectively;N was the negative control).

图8 草莓白粉病菌普通PCR扩增灵敏度的检测结果

Fig.8 Detection results of sensitivity of strawberry powdery mildew by using PCR amplification

图9 草莓白粉病菌 LAMP优化体系重复性检测结果 1.叶片(发病轻);2.果实(发病轻);3.果柄(发病较轻);4.叶片(发病严重);5.果实(发病严重);6.果柄(发病严重);7.健康果实。

Fig.9 Repeatability test results of lamp optimized system for strawberry powdery mildew 1.Strawberry blade (mild disease);2.Strawberry fruit (mild disease);3.Strawberry stalk(slightly affected);4.Strawberry blade (serious disease);5.Strawberry fruit (serious disease);6.Strawberry stalk(serious disease);7.Strawberry healthy fruit.

图10 草莓白粉病菌LAMP田间检测结果 1~7为草莓叶片样本:1.发病很轻叶片,2,3.发病较重,4,5.发病严重,6,7.健康叶片;8~14.草莓果实样本:8,9.发病较轻,10.发病较重,11,12.发病严重,13,14.健康果实;15~21.草莓果柄样本:15.发病较轻,16,17.发病较重,18,19.发病严重,20,21.健康果柄。

Fig.10 Field detection results of strawberry powdery mildew by using LAMP 1—7.Strawberry leaf samples:1.Leaves with lighter disease,2,3.Leaves with more heavy disease,4,5.Leaves with serious disease,6,7.Healthy leaves;8—14.Strawberry fruit samples:8,9.Mild disease,10.More severe disease,11,12.Serious disease,13,14.Healthy fruits;15—21.Strawberry stalk samples:15.Stalk with light disease,16,17.Stalks with heavy disease,18,19.Stalks with serious disease,20,21.Healthy leaves.

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