直接多重TaqMan qPCR方法快速鉴定褐飞虱属3种飞虱

doi:10.16819/j.1001-7216.2023.220903

中国水稻科学 ›› 2023, Vol. 37 ›› Issue (3): 329-336.DOI: 10.16819/j.1001-7216.2023.220903

• 实验技术 • 上一篇

直接多重TaqMan qPCR方法快速鉴定褐飞虱属3种飞虱

罗举¹, 杨素文², 贝文勇³, 余军伟⁴, 唐健¹, 刘淑华¹^,^*()

¹中国水稻研究所，杭州 311401
²湖南省邵东市牛马司镇农业综合服务中心，湖南邵阳 422803
³广西昭平县植保植检站，广西贺州 546800
⁴杭州优思达生物技术有限公司，杭州 310015

收稿日期:2022-09-06 修回日期:2022-11-12 出版日期:2023-05-10 发布日期:2023-05-16
通讯作者: ^*email: liushuhua@caas.cn
基金资助:
浙江省自然科学基金资助项目(LGN21C140002);十四五重点研发计划资助项目(2021YFD1401100);水稻生物学国家重点实验室开放课题(20210303);中央级公益性科研院所基本科研业务费专项(CPSIBRF-CNRRI-202123)

Direct Multiplex TaqMan qPCR Assay for Rapid Detection of Three Sibling Species from Nilaparvata Distant

LUO Ju¹, YANG Suwen², BEI Wenyong³, YU Junwei⁴, TANG Jian¹, LIU Shuhua¹^,^*()

¹China National Rice Research Institute, Hangzhou 311401, China
²Agricultural Comprehensive Service Center, Niumashi Town, Shaodong City, Hunan Province, Shaoyang 422803, China
³Plant Protection and Inspection Station of Zhaoping County, Guangxi Zhuang Autonomous Region, Hezhou 546800, China
⁴Hangzhou Ustar Biotechnology Co., Ltd., Hangzhou 310015, China

Received:2022-09-06 Revised:2022-11-12 Online:2023-05-10 Published:2023-05-16
Contact: ^*email: liushuhua@caas.cn

摘要/Abstract

摘要：

【目的】褐飞虱（Nilaparvata lugens）是水稻重大害虫，灯光诱捕一直是褐飞虱监测的重要方法。然而，褐飞虱的两个同属近似种伪褐飞虱（N. muiri）和拟褐飞虱（N. bakeri）也具有扑灯行为，常被误判为褐飞虱。针对测报灯下褐飞虱属近似种形态鉴定费时费力、专业技能要求高，伪褐飞虱和拟褐飞虱常被误判为褐飞虱这一问题，拟建立一种褐飞虱属3种飞虱的快速分类鉴定方法。【方法】以条形码基因ITS1为靶标，筛选褐飞虱属通用引物及物种特异性探针，建立并优化直接多重Taqman qPCR检测体系（Direct Multiplex TaqMan quantitative PCR, dmTqPCR），并分析其特异性、灵敏度及实用性。【结果】本研究建立的3种飞虱dmTqPCR检测方法特异性强，可准确区分褐飞虱、伪褐飞虱和拟褐飞虱；灵敏度高，检出限可达10拷贝/反应；大样本检测结果表明，382个样本从样本获取到结果输出的整个过程可在3 h内完成，检出率及准确率均为100%。【结论】本研究建立的褐飞虱属近似种的dmTqPCR鉴定方法可以用于褐飞虱、伪褐飞虱和拟褐飞虱的快速分类鉴定，有利于灯下褐飞虱的精准测报。

关键词: 褐飞虱, 伪褐飞虱, 拟褐飞虱, ITS1, 直接多重Taqman qPCR

Abstract:

【Objective】Nilaparvata lugens is a destructive insect pest on rice, and light trapping has always been an important monitoring method. However, its two sibling species, N. muiri and N. bakeri, can also be trapped by light and are easily mistaken as N. lugens. Morphological identification of N. lugens and its sibling species is time-consuming and labor-intensive, with the requirement for expertise and experience. To address this problem for identification of N. lugens and its two sibling species, the present study is aimed to develop a rapid and accurate identification method.【Method】At first, design and select the interspecies general primes and species-specific probes based on the barcoding gene ITS1. Then, establish and optimize the Direct Multiple Taqman qPCR assay (dmTqPCR). Finally, analyze and evaluate the specificity, sensitivity and practicability.【Results】The dmTqPCR assay developed in the present study has high specificity and high sensitivity, which could accurately identify N. lugens, N. bakeri and N. muiri, and the LOD (limit of detection) are up to 10 copies/reaction. The entire detection process for 382 specimens, including the crude tissue liquid preparation, amplification and detection, can be completed in 180 min with an accuracy of 100%.【Conclusion】The established dmTqPCR assay is suitable for rapid and accurate identification of N. lugens, N. muiri and N. bakeri, and it is expected to promote the development of precision prediction and forecasting of N. lugens.

Key words: Nilaparvata lugens, Nilaparvata muiri, Nilaparvata bakeri, ITS1, direct multiple Taqman qPCR

罗举, 杨素文, 贝文勇, 余军伟, 唐健, 刘淑华. 直接多重TaqMan qPCR方法快速鉴定褐飞虱属3种飞虱[J]. 中国水稻科学, 2023, 37(3): 329-336.

LUO Ju, YANG Suwen, BEI Wenyong, YU Junwei, TANG Jian, LIU Shuhua. Direct Multiplex TaqMan qPCR Assay for Rapid Detection of Three Sibling Species from Nilaparvata Distant[J]. Chinese Journal OF Rice Science, 2023, 37(3): 329-336.

图/表 7

图1 褐飞虱及其两个近似种的ITS1基因多序列比对 Nl―褐飞虱；Nm―伪褐飞虱；Nb―拟褐飞虱。下划线序列代表引物位置，蓝色背景为探针位置。

Fig. 1. Multiple alignment of ITS1 genes from N. lugens and its two sibling species. Nl, N. lugens; Nm, N.muiri; Nb, N. bakeri. Primer sequences were underlined, and the probe sequences were highlighted with blue background.

表1 本研究用到的引物和探针

Table 1. Primers and probes used in the study.

引物/探针名称 Primer/Probe name	引物/探针序列（5’-3’） Primer/Probe sequence (5’-3’)	描述 Description
ITS1-F	CCAAGTGATCCACCACTCAGG	褐飞虱属通用引物General primer
ITS1-R	GGATTCGTCTTGTCGGCTAGG	褐飞虱属通用引物General primer
Nl-ITS1-P	FAM-CCACCATACATTGTAATG-MGB	褐飞虱探针N.1ugens-specific probe
Nm-ITS1-P	VIC-CTAGTCATTACACAATGTA-MGB	伪褐飞虱探针N.muiri-specific probe
Nb-ITS1-P	ROX-CATTACATTGTATGTTGGC-MGB	拟褐飞虱探针N.bakeri-specific probe

表2 样本收集地点

Table 2. Regions of specimen collection in China.

地点 Location	坐标Coordinates		样本数量Number of specimen
地点 Location	东经 Longitude (E)	北纬 Latitude (N)	褐飞虱 N. lugens	伪褐飞虱 N. muiri	拟褐飞虱 N. bakeri
广西Guangxi	110.80	24.17	85	59	21
湖南Hunan	111.73	27.25	75	27	43
浙江Zhejiang	119.95	30.07	29	35	8

图2 褐飞虱属通用引物ITS1-F/R的特异性验证 1―水；2~3―褐飞虱；4~5―伪褐飞虱；6~7―拟褐飞虱；8―白背飞虱；9―灰飞虱；10―烟翅白背飞虱；11―稗飞虱；12―廖飞虱；13―短头飞虱；14―黑边梅塔飞虱；M―100 bp DNA标准品

Fig. 2. Specificity analysis of the inter-speies general primes ITS1-F/R. 1, H2O; 2－3, N. lugens; 4－5, N. muiri; 6－7, N. bakeri; 8, S. furcifera; 9, L. striatellus; 10, S. kolophon; 11, S. longifurcifera; 12, H. gayasana; 13, E. nawaii; 14, M. propinqua; M, 100 bp DNA marker.

图3 直接多重TaqMan qPCR体系的特异性试验 Nl―褐飞虱；Nm―伪褐飞虱；Nb―拟褐飞虱。

Fig. 3. Specificity analysis of the direct multiplex TaqMan qPCR assay. Nl, N. lugens; Nm, N.muiri; Nb, N. bakeri.

图4 直接多重TaqMan qPCR灵敏性测试试验 1~8分别代表1.0×107、1.0×106、1.0×105、1.0×104、1.0×103、1.0×102、1.0×101、1.0×100 拷贝/μL。

Fig. 4. Sensitivity analysis of the direct multiplex TaqMan qPCR assay. 1－8 represent 1.0×107, 1.0×106, 1.0×105, 1.0×104, 1.0×103, 1.0×102, 1.0×101, 1.0×100 copies /μL, respectively.

表3 直接多重TaqMan qPCR检测体系对382个样本检测的部分结果

Table 3. Partial dmTqPCR assay results for the 382 Nilaparvata species individuals.

样品编号 Sample No.	形态鉴定 Morphological identification	Ct值Cycle of threshold
样品编号 Sample No.	形态鉴定 Morphological identification	Nl 探针 Nl probe	Nm 探针 Nm probe	Nb 探针 Nb probe
1	Nl	24.91	Un	Un
2	Nm	Un	26.72	Un
3	Nl	25.50	Un	Un
4	Nb	Un	Un	24.68
5	Nm	Un	26.75	Un
6	Nm	Un	27.78	Un
7	Nl	26.33	Un	Un
8	Nb	Un	Un	24.28
9	Nm	Un	27.34	Un
10	Nl	25.46	Un	Un
11	Pc	21.80	21.75	22.12
12	Nc	Un	Un	Un

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直接多重TaqMan qPCR方法快速鉴定褐飞虱属3种飞虱

Direct Multiplex TaqMan qPCR Assay for Rapid Detection of Three Sibling Species from Nilaparvata Distant

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