Visual detection method of Colletotrichum scovilleiin fruit and vegetable based on LAMP

doi:10.3969/j.issn.1004-1524.2022.01.12

Acta Agriculturae Zhejiangensis ›› 2022, Vol. 34 ›› Issue (1): 98-103.DOI: 10.3969/j.issn.1004-1524.2022.01.12

• Plant Protection • Previous Articles Next Articles

Visual detection method of Colletotrichum scovilleiin fruit and vegetable based on LAMP

LUO Yue^a^,^b(), WU Xiaomao^a^,^b^,^*(), LIU Ali^a^,^b, YAO Xiaolong^a^,^b, LI Rongyu^a^,^b, GENG Guangdong^b

a. Institute of Crop Protection
b.College of Agriculture, Guizhou University, Guiyang 550025, China

Received:2020-12-24 Online:2022-01-25 Published:2022-02-05
Contact: WU Xiaomao

Abstract

Abstract:

To provide an efficient, convenient, specific and visual detection method for the detection of Colletotrichum scovillei of fruit and vegetable, taking the ITS sequence of the internal transcription spacer of C.scovillei of fruit and vegetable as the target gene, 4 LAMP primers and 1 loop primer were designed. By choice the primer ratio, reaction time, reaction system and other conditions, a LAMP detection method with specific amplification for C. scovillei was established. The results of the specificity test show that the established LAMP reaction system was amplified for 60 min at the optimal reaction temperature of 65 ℃. The reaction system using C. scovillei DNA as a template was used as the indicator calcein and hydroxynaphthol. Under the action of blue, green and blue appear, respectively. The reaction result was positive, and the amplified product was detected as a ladder band by 2% agarose gel electrophoresis. The reaction system and blank control using the DNA of other 8 control strains as templates,they were orange and purple respectively, and both were negative reactions, and no amplified bands appeared, indicating that the LAMP detection method is highly specific to C.scovillei of fruit and vegetable.

Key words: LAMP, fruit and vegetable, Colletotrichum scovillei, visual detection

CLC Number:

S432.9

LUO Yue, WU Xiaomao, LIU Ali, YAO Xiaolong, LI Rongyu, GENG Guangdong. Visual detection method of Colletotrichum scovilleiin fruit and vegetable based on LAMP[J]. Acta Agriculturae Zhejiangensis, 2022, 34(1): 98-103.

Figures/Tables 5

Table 1 Primer sequence

引物名称 Primer name	序列 Sequence(5'→ 3')
F3	AGCATTCTGGCGAGCATG
B3	TGATCCGAGGTCAACCTGTA
FIP	CCTTTAAGGGCCCACGTGTGCAGCGTCATTT-CAACCCTCAA
BIP	CGGAGCCTCCTTTGCGTAGTAAGGGTTTTAC-GGCAAGAGTCC
LB	CTAACGTCTCGCACTGGGAT

Fig.1 Amplification results of LAMP system at different reaction temperature and time M:DL2 000 marker. a: Amplification results of LAMP system at different reaction temperatures; 1, negative control; 2, 60 ℃;3, 62 ℃; 4, 64 ℃; 5, 65 ℃; 6, 66 ℃.b: Amplification result of LAMP system with different reaction times; 1, negative control; 2,30 min; 3,40 min; 4,50 min; 5,60 min; 6, 70 min.

Fig.2 LAMP reaction system for detecting Colletotrichum scovillei a: LAMP product agarose gel electrophoresis detection results; b: LAMP product hydroxynaphthol blue color results; c: LAMP product calcein fluorescence color results. 1, negative control (-); 2, Colletotrichum scovillei (+);3, Colletotrichum scovillei (+); 4, negative control (-).

Fig.3 Specificity of LAMP method under the indication of calcei M: DL2000 DNA marker。a: LAMP product calcein fluorescence coloring result; b: LAMP product agarose gel electrophoresis detection result. 1, Negative control; 2-4, Colletotrichum scovillei; 5, Colletotrichum gloeosporioides; 6-8, Colletotrichum karstii; 9-10, Magnaporthe oryzae; 11-12, Aclinonema sp.

Fig.4 Specificity of LAMP method under the indication of hydroxynaphthol blue M: DL2000 DNA marker。 a: LAMP product hydroxynaphthol blue color result; b: LAMP product agarose gel electrophoresis detection result. 1, Negative control; 2-4,Colletotrichum scovillei; 5, Colletotrichum gloeosporioides; 6-8, Colletotrichum karstii; 9-10, Magnaporthe oryzae; 11-12,Aclinonema sp.

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Visual detection method of Colletotrichum scovilleiin fruit and vegetable based on LAMP

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