Loop-mediated Isothermal Amplification Assay for Rapid Diagnosis of Aphelenchoides besseyi

doi:10.16819/j.1001-7216.2017.7001

Abstract

Abstract:

【Objective】 This study was performed to provide a new method for the rapid detection and early diagnosis of Aphelenchoides besseyi. 【Method】The loop-mediated isothermal amplification (LAMP) specific primers of A. besseyi were designed based on the nematode 18S ribosomal RNA gene, including two external primer, two inner primer and two loop primer. The DNA sample was amplified by LAMP primers using isothermal amplification. The amplified products were detected by electrophoresis and fluorescent dye. It proved that there was A. besseyi in the test sample if there was clear DNA ladder in the electrophoresis results or it showed green fluorescence in the fluorescent dye results. 【Result】The results showed that LAMP primers could detect and identify single egg, juvenile, female and male of A. besseyi, and could also directly detect A. besseyi from multiple nematode species samples and plant tissue samples, the sensitivity of LAMP assay was 1/1000 of single nematode DNA. 【Conclusion】This diagnosis method has the advantages of accuracy, sensitivity, stability and intuitive results, and is also simple and more practical.

Key words: Aphelenchoides besseyi, LAMP, rapid diagnosis, sensitivity

摘要：

目的本研究旨在为水稻干尖线虫(Aphelenchoides besseyi Christie, 1942)的快速检测鉴定和早期诊断提供一个新的检测方法。方法利用水稻干尖线虫18S核糖体RNA基因,设计了水稻干尖线虫LAMP的特异引物,该引物包括1对外引物、1对内引物和1对环引物,以LAMP特异引物对待测样品DNA进行恒温扩增,扩增产物通过电泳法和荧光染料法进行检测,电泳检测出现阶梯状条带或加入荧光染料显现绿色荧光,则证明待检样品中含有水稻干尖线虫。结果本方法可以检测鉴定水稻干尖线虫不同虫态（雌虫、雄虫、幼虫或卵）的个体,可以从多种线虫混合的样品和植物组织样品中直接检测出水稻干尖线虫,检测灵敏度达到1/1000条虫DNA。结论本检测方法具有准确、灵敏、稳定和结果直观的优点,且操作简便、实用性强。

关键词: 水稻干尖线虫, LAMP, 快速检测, 灵敏度

CLC Number:

S435.112⁺.9

Zongshi BAI, Meng QIN, Lirong ZHAO, Yuchun HAN, Dongwei WANG, Chunling XU, Hui XIE. Loop-mediated Isothermal Amplification Assay for Rapid Diagnosis of Aphelenchoides besseyi[J]. Chinese Journal OF Rice Science, 2017, 31(4): 432-440.

白宗师, 秦萌, 赵立荣, 韩玉春, 王东伟, 徐春玲, 谢辉. 水稻干尖线虫的环介导恒温扩增技术(LAMP)快速检测方法[J]. 中国水稻科学, 2017, 31(4): 432-440.

Figures/Tables 8

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种群编号 Population	寄主 Host	地理来源 Geographical origin
HB	水稻Oryza sativa	海南省海口市狮子岭 Lion mountain, Haikou, Hainan
GB	水稻O. sativa	广东广州华南农业大学岑村 Cen Village, South China Agricultural University, Guangzhou, Guangdong
HM5	水稻O. sativa	广东惠州市马安镇龙潭管理区 Longtan District Management, Maan Town, Huizhou, Guangdong
HC6	水稻O. sativa	广东惠州市陈江镇洋岭村 Yangling Village, Chenjiang Town, Huizhou, Guangdong
A-1	水稻O. sativa	安徽淮南宫集镇 Gongji Town, Huainan, Anhui
YQ1	水稻O. sativa	贵州余庆县白泥镇董家冲村 Dongjiachong Village, Baini Town, Yuqing County, Guizhou
YQ3	水稻O. sativa	贵州余庆县白泥镇陆家寨村 Lujiazhai Village, Baini Town, Yuqing County, Guizhou
G7	水稻O. sativa	广东茂名高州市谢鸡镇义山村 Yishan Village, Xieji Town, Maoming, Gaozhou, Guangdong
G27	水稻O. sativa	广东茂名茂南区山阁镇 Shan’ge Town, Maonan District, Maoming, Guangdong
S24	草莓 Fragaria ananassa	深圳市龙岗区同乐基地 Tongle Base, Longgang District, Shenzhen
N10	水稻O. sativa	江苏南京市六合区雄州镇红星村 Hongxing Village, Xiongzhou Town, Liuhe District, Nanjing, Jiangsu
N28	水稻O. sativa	江苏南京市六合区横梁镇光明村 Guangming Village, Hengliang Town, Liuhe District, Nanjing, Jiangsu
N47	水稻O. sativa	江苏淮安市楚州区平桥镇沈庄村 Shenzhuang Village, Pingqiao Town, Chuzhou District, Huaian, Jiangsu
N51	水稻O. sativa	江苏淮安市楚州区平桥镇东风村 Dongfeng Village, Pingqiao Town, Chuzhou District, Huaian, Jiangsu
N54	水稻O. sativa	江苏淮安市楚州区平桥镇三万村 Sanwan Village, Pingqiao Town, Chuzhou District, Huaian, Jiangsu
N65	水稻O. sativa	江苏淮安市楚州区淮城镇刘迈村 Liumai Village, Huaicheng Town, Chuzhou District, Huaian, Jiangsu
N71	水稻O. sativa	江苏淮安市淮阴区袁集镇云洞村 Yundong Village, Yuanji Town, Huaiyin District, Huaian, Jiangsu
N80	水稻O. sativa	江苏淮安市淮阴区袁集镇袁集村 Yuanji Village, Yuanji Town, Huaiyin District, Huaian, Jiangsu
X5	水稻O. sativa	福建厦门翔安区新圩镇马塘村 Matang Village, Xinxu Town, Xiangan District, Xiamen, Fujian
X8	水稻O. sativa	福建厦门翔安区新圩镇 Xinxu Town, Xiangan District, Xiamen, Fujian
X10	水稻O. sativa	福建厦门翔安区新圩镇上宅村 Shangzhai Village, Xinxu Town, Xiangan District, Xiamen, Fujian
Xi	水稻O. sativa	福建厦门同安区五显镇西洋村 Xiyang Village, Wuxian Town, Tongan District, Xiamen, Fujian
S-2	水稻O. sativa	深圳市龙岗区 Longgang District, Shenzhen
HN-2	水稻O. sativa	河南省罗山县潘新乡李桥村 Liqiao Village, Panli Town, Luoshan County, Henan

种群编号 Population	寄主 Host	地理来源 Geographical origin
HB	水稻Oryza sativa	海南省海口市狮子岭 Lion mountain, Haikou, Hainan
GB	水稻O. sativa	广东广州华南农业大学岑村 Cen Village, South China Agricultural University, Guangzhou, Guangdong
HM5	水稻O. sativa	广东惠州市马安镇龙潭管理区 Longtan District Management, Maan Town, Huizhou, Guangdong
HC6	水稻O. sativa	广东惠州市陈江镇洋岭村 Yangling Village, Chenjiang Town, Huizhou, Guangdong
A-1	水稻O. sativa	安徽淮南宫集镇 Gongji Town, Huainan, Anhui
YQ1	水稻O. sativa	贵州余庆县白泥镇董家冲村 Dongjiachong Village, Baini Town, Yuqing County, Guizhou
YQ3	水稻O. sativa	贵州余庆县白泥镇陆家寨村 Lujiazhai Village, Baini Town, Yuqing County, Guizhou
G7	水稻O. sativa	广东茂名高州市谢鸡镇义山村 Yishan Village, Xieji Town, Maoming, Gaozhou, Guangdong
G27	水稻O. sativa	广东茂名茂南区山阁镇 Shan’ge Town, Maonan District, Maoming, Guangdong
S24	草莓 Fragaria ananassa	深圳市龙岗区同乐基地 Tongle Base, Longgang District, Shenzhen
N10	水稻O. sativa	江苏南京市六合区雄州镇红星村 Hongxing Village, Xiongzhou Town, Liuhe District, Nanjing, Jiangsu
N28	水稻O. sativa	江苏南京市六合区横梁镇光明村 Guangming Village, Hengliang Town, Liuhe District, Nanjing, Jiangsu
N47	水稻O. sativa	江苏淮安市楚州区平桥镇沈庄村 Shenzhuang Village, Pingqiao Town, Chuzhou District, Huaian, Jiangsu
N51	水稻O. sativa	江苏淮安市楚州区平桥镇东风村 Dongfeng Village, Pingqiao Town, Chuzhou District, Huaian, Jiangsu
N54	水稻O. sativa	江苏淮安市楚州区平桥镇三万村 Sanwan Village, Pingqiao Town, Chuzhou District, Huaian, Jiangsu
N65	水稻O. sativa	江苏淮安市楚州区淮城镇刘迈村 Liumai Village, Huaicheng Town, Chuzhou District, Huaian, Jiangsu
N71	水稻O. sativa	江苏淮安市淮阴区袁集镇云洞村 Yundong Village, Yuanji Town, Huaiyin District, Huaian, Jiangsu
N80	水稻O. sativa	江苏淮安市淮阴区袁集镇袁集村 Yuanji Village, Yuanji Town, Huaiyin District, Huaian, Jiangsu
X5	水稻O. sativa	福建厦门翔安区新圩镇马塘村 Matang Village, Xinxu Town, Xiangan District, Xiamen, Fujian
X8	水稻O. sativa	福建厦门翔安区新圩镇 Xinxu Town, Xiangan District, Xiamen, Fujian
X10	水稻O. sativa	福建厦门翔安区新圩镇上宅村 Shangzhai Village, Xinxu Town, Xiangan District, Xiamen, Fujian
Xi	水稻O. sativa	福建厦门同安区五显镇西洋村 Xiyang Village, Wuxian Town, Tongan District, Xiamen, Fujian
S-2	水稻O. sativa	深圳市龙岗区 Longgang District, Shenzhen
HN-2	水稻O. sativa	河南省罗山县潘新乡李桥村 Liqiao Village, Panli Town, Luoshan County, Henan