Duplex-RPA Detection for Bursaphelenchus xylophilus and Bursaphelenchus mucronatus

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

Abstract

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

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.

Figures/Tables 8

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

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	--

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	-	+

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

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

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

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

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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