RAA-DETECTOR布鲁氏菌核酸快速检测方法的建立及应用

doi:10.11843/j.issn.0366-6964.2025.10.031

Abstract

Abstract:

The purpose of this study is to establish efficient and rapid detection systems for brucellosis that does not rely on professional equipment and personnel, and finished rapid detection of clinical samples within 40 minutes. First, the genomes of different Brucellaspecies were compared, and the core consensus sequence of Brucella was found. The positive plasmid was constructed using the BrucellaS2 strain gene SEQ NO.6 (CN 105018489A) as a template for subsequent experiments; CRISPR/AsCas12a and LwaCas13a proteins were expressed by prokaryotic expression methods; the recombinase-mediated isothermal nucleic acid amplification technology (RAA) and in vitro expression technology combined with the AsCas12a/LwaCas13a DETECTOR system were used to optimize the efficient detection system and the detection was determined by the infinite dilution method. The designed RAA primers can effectively amplify DNA fragments in samples. Optimal DETECTOR systems obtained 200 nmol·L^-1 AsCas12a protein, 200 nmol·L^-1 gRNA; also needs 300 nmol·L^-1 LwaCas13a and 400nM crRNA. The optimal reaction time of the detection system of RAA-AsCas12a and RAA-LwaCas13a was 35 min, the optimal detection time of the lateral flow strip was 20 min, the minimum detection concentration of bacteria was 10 Copies ·μL^-1, and it had good detection specificity. The detection test of clinical samples showed both methods had good repeatability and detection accuracy, but LwaCas13a had higher precision, but there was no significant difference between the two methods. This study established two RAA-CRISPR/Cas Brucellanucleic acid rapid detection systems, which can be well applied in clinical, and the RAA-LwaCas13a detection method has higher accuracy.

Key words: Brucella, RAA, DETECTOR system, SHERLOCK system, AsCas12a, LwaLwaCas13a

CLC Number:

S852.614

ZHANG Yunlong, WANG Jinglei, ZHU Yajie, ZHANG Mingjie, KANG Ao, ZHOU Xiang, WEI Kai, CAO Hongfang, LI Qiang, WANG Yong, SU Feng. Establishment and Application of a Nucleic Acid Detection Method for Brucella based on RAA-DETECTOR System[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(10): 5115-5124.

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References 18

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名称 Name	序列(5′→3′) Sequence(5′→3′)	产物大小/bp Product size
PF1	TTCACCGTCATCGGATAATAGGTGCCA	562
PR1	GGCGGGCTGCATTCTTTACTCTGC
RAA-F1	CATCATTGCAGACGATCATGCATTCGCGGC	287
RAA-R1	CTTCGAGGCCAACCGCAAGGCCATGCTGGC
PF2	TAATACGACTCACTATAGGGTTCACCGTCATCGGATAATAGGTGCCA	582
PR2	GGCGGGCTGCATTCTTTACTCTGC
RAA-F2	TAATACGACTCACTATAGGGCATCATTGCAGACGATCATGCATTCGCGGC	307
RAA-R2	CTTCGAGGCCAACCGCAAGGCCATGCTGGC

名称 Name	序列(5′→3′) Sequence (5′→3′)
AsCas12a gRNA	UAAUUUCUACUAGUAGA GGCGCGCUUUGUACCGCCAG
LwaCas13a crRNA	GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAAC GGCUUCAUCACCGGCAUCGGGCGCGUAU

组分 Component	体积/μL Volume
Nuclease-free water	Up to 20
10× Reaction Buffer	2
ATP	2
GTP	2
UTP	2
CTP	2
Template DNA	2
T7 RNA Polymerase Mix (Cas13a only)	2
RAA-PF1/RAA-PR1 (RAA-PF2/RAA-PR2)	2
总体积Total volume	20

组分 Component	体积/μL Volume
AsCas12a protein	4
crRNA	2
ssDNA报告分子ssDNA reporter molecules	4
RAA扩增产物RAA amplification products	20
NEBuffer2.1	8
RNase inhibitor	2
总体积Total volume	40

组分 Component	体积/μL Volume
LwaCas13a protein	4
crRNA	2
ssRNA报告分子ssRNA reporter molecule	4
RAA-T7转录产物RAA-T7 transcripts	20
NEBuffer2.1	8
RNase inhibitor	2
总体积Total volume	40

Establishment and Application of a Nucleic Acid Detection Method for Brucella based on RAA-DETECTOR System

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sgRNA浓度/(nmol·L^-1) sgRNA concentration	AsCas12a蛋白浓度/(nmol·L^-1) AsCas12a protein concentration
sgRNA浓度/(nmol·L^-1) sgRNA concentration	50	100	200	300	400
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crRNA浓度/(nmol·L^-1) crRNA concentration	LwaCas12a蛋白浓度/(nmol·L^-1) LwaCas12a protein concentration
crRNA浓度/(nmol·L^-1) crRNA concentration	50	100	200	300	400
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项目Item	阳性Positive	阴性Negative	总和Total	正比率/% Positive ratio	准确性/% Accuracy
PCR	15	55	70	21.43	100
RAA-CRISPR-Cas12a	16	54	70	22.86	95.71
RAA-CRISPR-Cas13a	17	53	70	24.29	97.14

项目 Item	重复 Copies	组间重复性变化 Intra-batch variation	组内重复性变化 Inter-batch variation
RAA-AsCas12a	10⁸	1.06%	1.17%
	10⁶	2.63%	1.24%
	10⁴	3.76%	6.01%
RAA-LwaCas13a	10⁸	1.11%	1.28%
	10⁶	1.35%	1.14%
	10⁴	2.89%	9.33%

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