非洲猪瘟实时荧光RPA诊断方法建立及应用

doi:10.3864/j.issn.0578-1752.2022.01.016

摘要/Abstract

摘要：

【目的】非洲猪瘟（African swine fever,ASF）自2018年首次暴发于我国沈阳后,迅速扩散至全国,对我国养猪业造成了沉重打击。研究针对其病原非洲猪瘟病毒（African swine fever virus,ASFV）建立一种快速核酸检测技术,为及时发现、准确处理ASF疫情提供一种快速诊断方法。【方法】针对ASFV保守的B646L（p72）基因,设计并筛选合适的引物、探针组合,利用基于重组酶-聚合酶扩增（recombinase polymerase amplification,RPA）的实时荧光RPA方法,对反应体系、反应条件、样品处理步骤等进行优化;利用质控品,对检测方法的特异性和灵敏度进行评价。对1 009份临床样品进行检测,并利用OIE推荐的实时荧光定量PCR方法（qPCR）和病毒分离,进一步验证该方法的检测结果。【结果】筛选得到一套合适的引物、探针,建立了针对ASFV p72基因的实时荧光RPA检测方法,优化后的反应体系总体积为25 µL,在实时荧光定量PCR仪器上,最适反应条件为39℃ 10 s,39℃ 20 s,40个循环,扩增反应时间约20 min;室温裂解法可取代传统核酸提取方法作为本检测的样品处理方法;使用优化后的条件,可在30 min内实现样品处理、核酸扩增和结果判定的整个过程,特异性评价结果显示本方法对猪细小病毒（PPV）、伪狂犬病毒（PRV）、圆环病毒1型/2型（PCV1/2）、猪瘟病毒（CSFV）、猪繁殖与呼吸综合征病毒（PRRSV）样品扩增结果均为阴性;敏感性评价结果显示本方法对基因I型、II型、IX型ASFV的模拟样品均可检出,对II型ASFV阳性模拟血样品可检测至10 拷贝/µL,对已知阳性临床组织样品可检至1﹕10^3.0稀释度,与OIE推荐的实时荧光定量PCR（qPCR）方法的检测灵敏度一致。通过对1 009份临床样品进行检测,实时荧光RPA诊断方法检出17份阳性,其结果与qPCR方法完全一致;病毒分离获得17份阳性培养物。【结论】建立的实时荧光RPA核酸检测方法操作简便,耗时短,具有较高的灵敏度和特异性,为临床提供了一种新型、简单、特异、快速诊断非洲猪瘟的方法。

关键词: 非洲猪瘟病毒, 重组酶聚合酶扩增, 实时荧光RPA, 核酸检测, 诊断技术

Abstract:

【Objective】 After the first outbreak of African Swine Fever (ASF) in Shenyang, China in 2018, it has rapidly spread to the whole country, severely hitting the pig industry. This study aimed to establish an optimized nucleic acid testing technique for African Swine Fever Virus (ASFV), so as to provide a fast and accurate method for early diagnosis and accurate treatment of ASF outbreaks. 【Method】 Appropriate primers and probes were designed and screened for the conserved gene B646L (p72) of ASFV, and a real-time fluorescent RPA assay based on recombinase polymerase amplification (RPA) was established. The reaction system, reaction conditions and sample treatment steps were optimized. Specificity and sensitivity of the optimized detection method were evaluated by using quality controls. In addition, 1 009 clinical samples were tested by the optimized real-time RPA, after which the results were further confirmed by the real time PCR recommended by OIE and through virus isolation. 【Result】 A pair of primers-probe combinations was successfully screened, and a real-time fluorescence RPA for detection of ASFV p72 gene was developed. The total volume of optimized reaction system was 25 μL. The reaction conditions were set as 39℃ 10 s, 39℃ 20 s, 40 cycles on the fluorescence quantitative PCR instrument, and the whole amplification reaction needs about 20 min. The analysis method at room temperature could replace the traditional nucleic acid extraction method, thus the whole process of sample treatment, nucleic acid amplification and result reading could be completed in 30 min. Specific evaluation showed that the real-time RPA was negative for porcine parvovirus (PPV), pseudorabies virus (PRV), circovirus type1/2 (PCV1/2), classical swine fever virus (CSFV) and porcine reproductive and respiratory syndrome virus (PRRSV); the sensitivity evaluation showed that the assay could detect type I/II/IX ASFV samples, and could detect 10 copies/μL of ASFV positive simulated blood samples and 1﹕10^3.0 dilution of positive clinical samples, which was as sensitive as the OIE-recommended qPCR method. Seventeen out of 1 009 clinical samples were tested positive using the real-time RPA, with the same results as by qPCR, 17 positive cultures were obtained from virus isolation. 【Conclusion】 A real-time RPA diagnostic method for ASF was developed, which was proved to be simple, less time consuming with high sensitivity and specificity, providing a new, simple, specific and rapid diagnostic method for ASF.

Key words: African swine fever virus, recombinase polymerase amplification, real-time fluorescent RPA, nucleic acid detection, diagnosis

张静远,缪发明,陈腾,李敏,扈荣良. 非洲猪瘟实时荧光RPA诊断方法建立及应用[J]. 中国农业科学, 2022, 55(1): 197-207.

ZHANG JingYuan,MIAO FaMing,CHEN Teng,LI Min,HU RongLiang. Development and Application of a Real-Time Fluorescent RPA Diagnostic Assay for African Swine Fever[J]. Scientia Agricultura Sinica, 2022, 55(1): 197-207.

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序号 Number	组分 Components	体积 Volume (μL)
1	Primer F	2.1
2	Primer R	2.1
3	Exo Probe	0.6
4	Rehydration buffer	29.5
5	Water	11.2
6	Template	1	1
7	MgOAc（280 mmol·L^-1）	1.25	1.25
	合计Total	25	25

体系A System A			体系B System B
组分 Components	体积 Volume(μL)		组分 Components	体积 Volume(μL)
Primer F	2.1		Rehydration Mix	40（2 reactions）
Primer R	2.1
Exo Probe	0.6
Rehydration buffer	29.5
Water	11.2
将溶解后的反应体系等分至两个反应管 Equal distribution of the dissolved amplification system into two tubes
Template	1	1	Template	1	1
MgOAc（280 mmol·L^-1）	1.25	1.25	MgOAc-B	4	4
合计 Total	25	25	合计 Total	25	25

名称 Name	序列（5′-3′）Sequence (5′-3′)
EXO-F	TAATAGCAGATGCCGATACCACAA
EXO-R	TTACATACCCTTCCACTACGGAGGC
EXO-P	GTCCCAACTAATATAAAATTCTCTTGCTC/i6FAMdT/G/idSp/A/iBHQ1dT/ACGTTAATATGACCAC-P

处理方法 Treatment	所需温度 Temperature	操作步骤 Operation Steps	处理时间 Time costs	Ct值Ct Value (2Ct≈1min)
处理方法 Treatment	所需温度 Temperature	操作步骤 Operation Steps	处理时间 Time costs	N	WP	SP
无处理Untreatment	/	/	0 min	/	33.92	19.39
磁珠法 Magnetic beads extraction	RT	结合,清洗,洗脱 Combination, Wash, Elution	10 min	/	22.23	17.76
柱提取法 Column extraction	RT	裂解,沉淀,离心,洗脱 Lysis, Precipitation, Centrifugation, Elution	30 min	/	23.54	14.02
裂解法 Lysis	RT	裂解,离心 Lysis, Centrifugation	5 min	/	24.07	14.25
煮沸法 Boiling	100℃,4℃	煮沸,冷藏,离心 Boiling, Refrigeration, Centrifugation	10 min	/	22.05	16.96

样品种类 Samples	稀释倍数 Dilutions	检测结果Results
		实时RPA Real-time RPA		荧光定量PCR rt-PCR
		Ct值Ct value	判定Read	Ct值Ct value	判定Read
脾脏 Spleen	1:10^1.0	30.49	+	28.00	+
	1:10^2.0	35.55	+	30.23	+
	1:10^3.0	38.35	+	34.71	+
	1:10^4.0	/	-	/	-
淋巴结 Lymph node	1:10^1.0	28.58	+	29.44	+
	1:10^2.0	34.61	+	30.11	+
	1:10^3.0	39.33	+	37.21	+
	1:10^4.0	/	-	/	-
血清 Serum	1:10^1.0	24.76	+	27.32	+
	1:10^2.0	29.68	+	32.33	+
	1:10^3.0	32.12	+	35.56	+
	1:10^4.0	/	-	/	-