Development of One-Step Nested-Locked Nucleic Acid Real-Time PCR Detection Method for African Swine Fever Virus

doi:10.11924/j.issn.1000-6850.casb2022-0055

Abstract

Abstract:

In this study, a highly sensitive and specific detection method was established based on the B646L (P72) protein gene of African swine fever virus (ASFV). The nested primers and probes of LNA-OSN-PCR (One Step Nested-Locked Nucleic Acid real-time PCR) were designed with Oligo 7 software; external primers were modified with locked nucleic acids. The reaction system and conditions of LNA-OSN-PCR were established and optimized, and the standard curve of LNA-OSN-PCR was established. Then, the sensitivity, specificity and repeatability of LNA-OSN-PCR were tested. A total of 96 clinical suspected diseased samples were detected by LNA-OSN-PCR and compared with the samples detected by conventional fluorescent PCR method. The reaction conditions and system of LNA-OSN-PCR were established and optimized, and the standard curve of LNA-OSN-PCR had good linearity (R²=0.9945). The LNA-OSN-PCR had a minimum detection limit of 3×10^-1 copies/μL, with a coefficient of variation of less than 3% and no cross-reaction with other viral or bacterial nucleic acids. Compared with the qPCR method recommended by OIE, the detection sensitivity of the LNA-OSN-PCR method was increased by 10-100 times. Among the 96 samples tested by LNA-OSN-PCR, 55 were positive and 41 were negative, with a higher positive detection rate than traditional qPCR and a better typical amplification curve. By combining the one-step nested PCR and lock-in nucleic acid modification, the LNA-OSN-PCR method for ASFV was established to provide a highly sensitive, specific and economical method for ASFV detection.

Key words: African swine fever virus, B646L (P72) protein gene, locked nucleic acid modified, one-step nested real-time PCR

SHI Keda, XU Minsheng, LI Yan, ZHANG Kunli, ZHAI Shaolun, GOU Hongchao, SONG Shuai, YANG Dongxia, ZANG Ying'an, SUN Mingfei, LI Chunling. Development of One-Step Nested-Locked Nucleic Acid Real-Time PCR Detection Method for African Swine Fever Virus[J]. Chinese Agricultural Science Bulletin, 2023, 39(11): 143-151.

Figures/Tables 8

References 28

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	引物和探针	序列(5’-3’)
LNA-OSN-PCR	内引物F1	ATAGATGAACATGCGTCTGG
	内引物R1	CAAAATCCTCATCAACACCG
	外引物F2	TG(+)^*GTATTCC(+)TCCC(+)GTG(+)GC(+)TTC
	外引物R2	C(+)CCCAGT(+)AGA(+)CGC(+)AATA(+)TACGC
	探针	FAM-CTGAAAGCTTATCTCTGCGTGGT-BHQ1
荧光定量PCR	VP72-F1	GCTTTCAGGATAGAGATACAGCTCT
	VP72-R1	CCGTAGTGGAAGGGTATGTAAGAG
	VP72-T1	FAM-CCGTAACTGCTCATGGTATCAATCTTATCG-BHQ1

	引物和探针	序列(5’-3’)
LNA-OSN-PCR	内引物F1	ATAGATGAACATGCGTCTGG
	内引物R1	CAAAATCCTCATCAACACCG
	外引物F2	TG(+)^*GTATTCC(+)TCCC(+)GTG(+)GC(+)TTC
	外引物R2	C(+)CCCAGT(+)AGA(+)CGC(+)AATA(+)TACGC
	探针	FAM-CTGAAAGCTTATCTCTGCGTGGT-BHQ1
荧光定量PCR	VP72-F1	GCTTTCAGGATAGAGATACAGCTCT
	VP72-R1	CCGTAGTGGAAGGGTATGTAAGAG
	VP72-T1	FAM-CCGTAACTGCTCATGGTATCAATCTTATCG-BHQ1

P72蛋白标准质粒/ (copies/μL)	组内		组间(Ct值)		置信区间(95%)
P72蛋白标准质粒/ (copies/μL)	$\bar{x}±s$ (Ct值)	变异系数/%	$\bar{x}±s$ (Ct值)	变异系数/%	置信区间(95%)
3×10³	18.56±0.12	0.62	18.79 ± 0.46	2.45	1.84~18.9
3×10²	21.51±0.10	0.48	21.67 ± 0.64	2.95	21.3~21.9
3×10¹	24.84±0.05	0.21	24.97 ± 0.48	1.90	24.7~25.1
3×10⁰	27.83±0.19	0.67	28.21 ± 0.14	0.50	27.9~28.2
3×10^-1	30.08±0.06	0.21	30.66 ± 0.77	2.50	29.9~30.8

P72蛋白标准质粒/ (copies/μL)	组内		组间(Ct值)		置信区间(95%)
P72蛋白标准质粒/ (copies/μL)	$\bar{x}±s$ (Ct值)	变异系数/%	$\bar{x}±s$ (Ct值)	变异系数/%	置信区间(95%)
3×10³	18.56±0.12	0.62	18.79 ± 0.46	2.45	1.84~18.9
3×10²	21.51±0.10	0.48	21.67 ± 0.64	2.95	21.3~21.9
3×10¹	24.84±0.05	0.21	24.97 ± 0.48	1.90	24.7~25.1
3×10⁰	27.83±0.19	0.67	28.21 ± 0.14	0.50	27.9~28.2
3×10^-1	30.08±0.06	0.21	30.66 ± 0.77	2.50	29.9~30.8

非洲猪瘟检测	一步法巢式锁核酸荧光定量PCR (LNA-OSN-PCR)	荧光探针PCR
阳性^*/份	55	47
阴性/份	41	49
合计/份	96	96
阳性检出率	57.3%	49.0%