李属坏死环斑病毒RT-RAA-CRISPR/Cas12a可视化检测方法的建立与应用

doi:10.3864/j.issn.0578-1752.2025.12.008

Abstract

Abstract:

【Objective】The study aims to establish a novel visual detection technique for prunus necrotic ringspot virus (PNRSV) by combining reverse transcription recombinase-aided amplification (RT-RAA) with CRISPR/Cas12a system (RT-RAA-CRISPR/ Cas12a).【Method】Primers with high amplification efficiency and strong specificity were designed and selected based on the conserved regions of the coat protein (CP) gene of PNRSV. The detection conditions, including primer, probe concentration, temperature, and reaction time were optimized to develop a visual detection method for PNRSV by RT-RAA-CRISPR/Cas12a technology. The specificity of this method was evaluated by detecting PNRSV and common Prunus viruses, including plum pox virus (PPV), apple mosaic virus (ApMV), cucumber mosaic virus (CMV), potato virus X (PVX), and potato virus Y (PVY). The total RNAs from PNRSV-infected fruit were diluted in 10-fold gradients, then RT-PCR, RT-RAA and RT-RAA-CRISPR/Cas12a were performed to compare the sensitivity of the three methods. The RT-RAA-CRISPR/Cas12a and RT-PCR methods were used to detect 31 peach fruit test samples suspected to be infected with the virus collected at the port to verify the practicability of the visual detection method.【Result】The RT-RAA-CRISPR/Cas12a-based visual detection method for PNRSV was successfully established. The optimized working concentrations were as follows: RT-RAA-PNRSV-F2/R2 primers at 0.4 μmol·L^-1, fluorescent reporter (FQ) at 800 nmol·L^-1, CRISPR-Cas12a at 200 nmol·L^-1, and PNRSV-crRNA (CRISPR RNA) at 240 nmol·L^-1, the reaction conditions were performed at 41 ℃ for 45 min. This method showed high specificity for PNRSV and had no cross-reaction with other common Prunus viruses. The limit of detection for PNRSV RNA in peach fruit samples reached 3.06 pg·μL^-1 and 306 fg·μL^-1 using RT-RAA and RT-RAA-CRISPR/Cas12a methods, respectively, showing the sensitivity of RT-RAA-CRISPR/Cas12a was 10 times higher than that of RT-RAA and RT-PCR. Among the 31 tested peach fruit samples at the port, 14 positive samples were identified by RT-PCR, while 15 positive samples were found by RT-RAA-CRISPR/Cas12a, indicating a high level of consistency between the two methods.【Conclusion】The RT-RAA-CRISPR/Cas12a visual detection method for PNRSV has been established. It is characterized by simplicity, rapidity, high sensitivity, high specificity, and visual readability, making it well-suited for rapid on-site detection of PNRSV.

Key words: prunus necrotic ringspot virus (PNRSV), RT-RAA, CRISPR/Cas12a, visual detection

ZHANG XiaoQi, SHEN JianGuo, LIAO FuRong, LI WeiMin, JIN YuJie, WUFUER Shayidan, ZHENG LuPing. Establishment and Application of RT-RAA-CRISPR/Cas12a-Based Visual Detection of Prunus Necrotic Ringspot Virus[J].Scientia Agricultura Sinica, 2025, 58(12): 2371-2381.

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References 27

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方法Method	引物名称Primer name	引物序列Primer sequence (5′ to 3′)	靶标长度Target size (bp)
RT-PCR	RT-PCR-PNRSV-F	TAGGGTTTCGAGCGGTATAGGA	525
RT-PCR	RT-PCR-PNRSV-R	TCATCGACCAGCAAGACATCAG	525
RT-RAA	RT-RAA-PNRSV-F1	GAATAACCCGAATAGGAATAGGAACCCGAATA	220
	RT-RAA-PNRSV-R1	CAACTGAGGGAGAGTGGTCGTGAAGTCAATAC	220
	RT-RAA-PNRSV-F2	CGAATAACCCGAATAGGAATAGGAACCCGA	310
	RT-RAA-PNRSV-R2	TTATAGTCCTCCACCATCCCAATCCAACCATT	310
	RT-RAA-PNRSV-F3	TATTGACTTCACGACCACTCTCCCTCAGTTGA	320
	RT-RAA-PNRSV-R3	CACTTACCACTACGTACAAATCCCTAACCAA	320
	RT-RAA-PNRSV-F4	GAGGTGACGACGACAGAGGCAGTGAAGTAC	352
	RT-RAA-PNRSV-R4	TTACCACTACGTACAAATCCCTAACCAAGACC	352

Establishment and Application of RT-RAA-CRISPR/Cas12a-Based Visual Detection of Prunus Necrotic Ringspot Virus

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试验组号 Group number	Cas12a浓度 Cas12a concentration (nmol·L^-1)	crRNA浓度 crRNA concentration (nmol·L^-1)	试验组号 Group number	Cas12a浓度 Cas12a concentration (nmol·L^-1)	crRNA浓度 crRNA concentration (nmol·L^-1)
1	400	480	9	400	240
2	300	480	10	300	240
3	200	480	11	200	240
4	100	480	12	100	240
5	400	360	13	400	120
6	300	360	14	300	120
7	200	360	15	200	120
8	100	360	16	100	120

试验组号 Group number	Cas12a浓度 Cas12a concentration (nmol·L^-1)	crRNA浓度 crRNA concentration (nmol·L^-1)	试验组号 Group number	Cas12a浓度 Cas12a concentration (nmol·L^-1)	crRNA浓度 crRNA concentration (nmol·L^-1)
1	400	480	9	400	240
2	300	480	10	300	240
3	200	480	11	200	240
4	100	480	12	100	240
5	400	360	13	400	120
6	300	360	14	300	120
7	200	360	15	200	120
8	100	360	16	100	120

试验组号 Group number	Cas12a浓度 Cas12a concentration (nmol·L^-1)	crRNA浓度 crRNA concentration (nmol·L^-1)	试验组号 Group number	Cas12a浓度 Cas12a concentration (nmol·L^-1)	crRNA浓度 crRNA concentration (nmol·L^-1)
1	400	480	9	400	240
2	300	480	10	300	240
3	200	480	11	200	240
4	100	480	12	100	240
5	400	360	13	400	120
6	300	360	14	300	120
7	200	360	15	200	120
8	100	360	16	100	120