松材线虫和拟松材线虫双重RPA检测研究

doi:10.13560/j.cnki.biotech.bull.1985.2021-0579

摘要/Abstract

摘要：

旨在建立一种以重组酶聚合酶扩增技术（RPA）为基础的快速检测方法,用于松材线虫和拟松材线虫的同步检测鉴定。根据松材线虫和拟松材线虫ITS区差异序列,分别设计松材线虫和拟松材线虫特异性上游引物Bx-rpa-F、Bm-rpa-F和两者通用下游引物Bxm-rpa-R。优化3条引物在反应体系中的最佳配比,建立双重RPA检测体系,并对其特异性和灵敏度进行分析。研究结果显示,双重RPA在37℃恒温条件反应30 min,即可完成对松材线虫和拟松材线虫核酸的同步扩增。Bx-rpa-F/Bm-rpa-F/Bxm-rpa-R对松材线虫的扩增片段为346 bp,对拟松材线虫的扩增产物为189 bp,且三者配比为5∶3∶8时,双重RPA扩增效果最好。双重RPA对松材线虫和拟松材线虫具有较高的检测特异性,对松材线虫的检测极限为10 pg/μL,相当于1/100条线虫,对拟松材线虫的检测极限为100 pg/μL,相当于1/10条线虫,其灵敏度低于常规PCR技术,但其满足对单条线虫检测的需求。双重RPA从松木样品中同步检测到松材线虫和拟松材线虫,操作简便、检测效率高、特异性强、灵敏度高、对仪器设备要求低,为松材线虫的检疫鉴定提供新方法。

关键词: 松材线虫, 重组酶聚合酶技术, 特异性, 灵敏度, 实用性

Abstract:

The aim of this work is to develop a rapid recombinase polymerase amplification（RPA）method for simultaneously detecting Bursaphelenchus xylophilus and Bursaphelenchus mucronatus. The forward RPA primer Bx-rpa-F,Bm-rpa-F and the common reverse RPA primer Bxm-rpa-R were designed according to the sequence variation of ITS from B. xylophilus and B. mucronatus. The concentration of the three RPA primers in duplex-RPA was optimized,duplex-RPA detection system was established,and its specificity and sensitivity were analyzed. The results showed that duplex-RPA simultaneously amplified DNA extracted from B. xylophilus and B. mucronatus at 37℃ within 30 min. The RPA product of B. xylophilus amplified with Bx-rpa-F/Bxm-rpa-R was 346 bp,and that of B. mucronatus with Bm-rpa-F/Bxm-rpa-R was 189 bp. In addition,duplex-RPA presented the best amplification efficiency when the concentration of Bx-rpa-F/Bm-rpa-F/Bxm-rpa-R was 5∶3∶8. Duplex RPA had high specificity and sensitivity for the detection of B. xylophilus and B. mucronatus. The detection limit of duplex-RPA for B. xylophilus was 10 pg/μL,which was equivalent to 1/100 single nematode;while the detection limit of duplex-RPA for B. mucronatus was 100 pg/μL,which was equivalent to 1/10 single nematode. The sensitivity was lower than that by conventional method,however,it met the requirement for detecting an individual nematode. In conclusion,duplex-RPA may simultaneously detect B. xylophilus and B. mucronatus from infected pine wood samples. Moreover,duplex-RPA has the advantages of simple operation,high detection efficiency,strong specificity,high sensitivity and low requirements for instruments and equipment,which provides a new method for quarantine and identification of B. xylophilus.

Key words: Bursaphelenchus xylophilus, recombinase polymerase amplification, specificity, sensitivity, practicability

方圆, 吴迅, 林宇, 王海燕, 吴慧平, 鞠玉亮. 松材线虫和拟松材线虫双重RPA检测研究[J]. 生物技术通报, 2021, 37(7): 183-190.

FANG Yuan, WU Xun, LIN Yu, WANG Hai-yan, WU Hui-ping, JU Yu-liang. Duplex-RPA Detection for Bursaphelenchus xylophilus and Bursaphelenchus mucronatus[J]. Biotechnology Bulletin, 2021, 37(7): 183-190.

图/表 8

表1 供试线虫种群

Table 1 Population information of nematodes

种类Species	种群 Population	来源 Origin
松材线虫B. xylophilus	Bxah1	中国安徽 Anhui,China
	Bxah2	中国安徽 Anhui,China
	Bxah3	中国安徽 Anhui,China
	Bxjs	中国江苏 Jiangsu,China
	Bxus	美国 USA
拟松材线虫B. mucronatus	Bmah1	中国安徽 Anhui,China
	Bmah2	中国安徽 Anhui,China
	Bmis	中国江苏 Jiangsu,China
	Bmsk	韩国South Korea
豆伞滑刃线虫B. doui	Bdsk	韩国 South Korea
食菌伞滑刃线虫B. fungivous	Bfsk	韩国 South Korea
莱奴尔夫滑刃线虫B. rainulfi	Brsk	韩国 South Korea
菊花滑刃线虫A. ritzemabosi	Arus	美国 USA
水稻干尖线虫A. besseyi	Abah1	中国安徽 Anhui,China
水稻干尖线虫A. besseyi	Abah12	中国安徽 Anhui,China

表2 用于双重RPA的引物及其序列

Table 2 Primers and their sequences for duplex-RPA

引物 Primer	序列 Sequence（5'-3'）	长度Length/bp	扩增长度 Amplification length/bp
Bx-rpa-F	TTCGTGCTCGTCACGATGATGCGATTGGTGACT	33	346
Bm-rpa-F	AAGTCTGGGTTTCTATGCGCTGCGTTGAGTCGA	33	189
Bxm-rpa-R	CGCAATTCACTGCGTTCTTCATCGACCCGCGAG	33	--

表3 松木样本中松材线虫与拟松材线虫的双重RPA检测

Table 3 Duplex-RPA detection for B. xylophilus and B. mucronatus

编号 Code	采集地点 Collecting location	双重RPA检测结果 Results from duplex-RPA detection
编号 Code	采集地点 Collecting location	B. xylophilus	B. mucronatus
AN01	安徽南陵 Nanling,Anhui	+	-
AN02	安徽南陵 Nanling,Anhui	+	-
AN03	安徽南陵 Nanling,Anhui	+	+
AC01	安徽滁州 Chuzhou,Anhui	+
AC02	安徽滁州 Chuzhou,Anhui	+	+
AC03	安徽滁州 Chuzhou,Anhui	+	+
AT01	安徽桐城Tongcheng,Anhui	+	-
AT02	安徽桐城 Tongcheng,Anhui	-	+
AT03	安徽桐城 Tongcheng,Anhui	-	+

图1 松材线虫和拟松材线虫双重RPA检测 1：松材线虫;2：拟松材线虫;3：松材线虫与拟松材线虫混合样本;4：阴性对照;M：DNA marker DL2000

Fig. 1 Duplex-RPA assay for detecting B. xylophilus and B. mucronatus 1：B. xylophilus. 2：B. mucronatus. 3：Mixed sample of B. xylophilus and B. mucronatus. 4：Negative control. M：DNA marker DL2000

图2 不同引物配比下的扩增产物 1-9：Bx-rpa-F、Bm-rpa-F和Bxm-rpa-R的添加量分别为2.4:0:2.4、2.1:0.3:2.4、1.8:0.6:2.4、1.5:0.9:2.4、1.2:1.2:2.4、0.9:1.5:2.4、0.6:1.8:2.4、0.3:2.1:2.4、0:2.4:2.4 μL;M：DNA marker DL2000

Fig. 2 Amplified products with different primer ratios 1-9：The contents of Bx-rpa-F,Bm-rpa-F and Bxm-rpa-R were 2.4:0:2.4,2.1:0.3:2.4,1.8:0.6:2.4,1.5:0.9:2.4,1.2:1.2:2.4,0.9:1.5:2.4,0.6:1.8:2.4,0.3:2.1:2.4,0:2.4:2.4 μL,respectively. M：DNA marker DL2000

图3 双重RPA特异性检测

Fig. 3 Specificity test of duplex-RPA 1：B. xylophilus Bxah1;2：B. xylophilus Bxah2;3：B. xylophilus Bxah3;4：B. xylophilus Bxjs;5：B. xylophilus Bxus;6：B. mucronatus Bmah1;7：B. mucronatus Bmah2;8：B. mucronatus Bmis;9：B. mucronatus Bmsk;10：B. doui Bdsk;11：B. fungivous Bfsk;12：B. rainulfi Brsk;13：A. ritzemabosi Arus;14：A. besseyi Abah1;15：A. besseyi Abah2

图4 双重RPA灵敏度检测 A：双重RPA与常规PCR对松材线虫的检测灵敏度;B：双重RPA与常规PCR对拟松材线虫的检测灵敏度;C：双重RPA对松材线虫与拟松材线虫DNA等比例混合样品的检测灵敏度。1-6：10-1、10-2、10-3、10-4、10-5、10-6条线虫;M：DNA marker DL2000

Fig. 4 Sensitivity test of duplex-RPA A：Sensitivity of duplex-RPA and conventional PCR in the detection of B. xylophilus. B：Sensitivity of duplex-RPA and conventional PCR in the detection of B. mucronatus. C：Sensitivity of duplex-RPA in the detection of B. xylophilus and B. mucronatus in equal proportion.1-6：10-1,10-2,10-3,10-4,10-5,and 10-6 of a single nematode.M：DNA marker DL2000

图5 松木样品中松材线虫和拟松材线虫双重RPA检测 1-9：AN01、AN02、AN03、AC01、AC02、AC03、AT01、AT02、AT03 RPA检测结果;10：阴性对照;M：DNA marker DL2000

Fig. 5 Duplex-RPA assay for detecting B. xylophilus and B. mucronatus from wood samples 1-9：RPA test results of AN01, AN02, AN03, AC01, AC02, AC03, AT01, AT02 and AT03;10：negative control;M：DNA marker DL2000

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