外源性马立克氏病病毒荧光定量PCR检测方法的建立

doi:10.3864/j.issn.0578-1752.2023.20.016

Abstract

Abstract:

【Objective】 In order to solve the problems of low sensitivity, long detection time, and poor discrimination of existing exogenous Marek’s disease virus (MDV) testing methods, this study was designed to establish two real-time PCR detection methods for the identification of MDV serotype 1 (MDV 1) and MDV serotype 3 (MDV 3) strains, which could be used for purity control of poultry-derived biological products. 【Method】 The UL19 sequences of MDV 1, MDV serotype 2 (MDV 2) and MDV 3 strains were downloaded from the NCBI database and were used for nucleotide and amino acid homology comparison. A pair of specific primers and corresponding Taqman probe was designed from the known sequence of conserved UL19 of MDV 1 CVI988 strain and MDV 3 FC126 strain, respectively, and two real-time PCR detection methods were established. The corresponding recombinant plasmids were constructed and used as positive standards to make standard curves, and the sensitivity of gene copy number of the methods were evaluated. Other avian virus-associated biological products, virus, the full-length plasmid of UL19 of MDV 2 SB-1 strain and the raw materials for production (SPF chicken embryo allantoic fluid, embryonic body, allantoic membrane, chicken embryo fibroblasts) were detected to evaluate the specificity of the established methods. 600, 60, 6, 0.6, 0.06, 0.006 and 0.0006 PFU of CVI988 or FC126 strains were detected, respectively, and the sensitivity of the established two methods for detecting live virions was evaluated. Three repeatability tests were performed using corresponding recombinant plasmids of different dilutions, and the correlation coefficient were calculated to analyze the reproducibility of the two established detection methods. 【Result】 The nucleotide and the derived amino acid homology of MDV UL19 in the same serotype was highly conserved with 99.99%, and the nucleotide homology between different serotypes was only about 75%, while the derived amino acid homology was only about 85%. MDV 1 and MDV 3 real-time PCR detection methods were established, respectively. About the MDV 1 real-time PCR detection method, the amplification efficiency was 98.8%, the correlation coefficient was 0.992, with the standard curve : Y=-3.351X+38.828 (Y = Ct, X = lg ( copy number)). About the MDV 3 real-time PCR detection method, the amplification efficiency was 95%, the correlation coefficient was 0.998, and the standard curve: Y=-3.447X+36.496 (Y = Ct, X = lg ( copy number)). The established detection methods could specially detect MDV 1 or MDV 3 without detecting any other avian virus-associated biological products, virus, the full-length plasmid of UL19 of MDV 2 SB-1 strain, along with production materials for poultry. The sensitivity of MDV 1 real-time PCR detection method was high, with the gene copy number detection limit of 32.8 copies/μL, which could detect at least 0.006 PFU of CVI988 strain. The sensitivity of MDV 3 real-time PCR detection method was high, with the gene copy number detection limit of 10 copies/μL, which could detect at least 0.006 PFU of FC126 strain. The coefficient of variation of the repeatability test was less than 1% in MDV 1 real-time PCR detection method, and less than 1.5% in MDV 3 real-time PCR detection method, respectively. 【Conclusion】 The established real-time PCR detection methods would be beneficial for detecting exogenous MDV 1 and MDV 3 strains in poultry-derived biological products.

Key words: Marek’s disease virus, real-time PCR, extraneous virus, detection

SU Jia, ZHAO Wei, LIU Dan, WANG Jia, BAI HongXu, WU HuaWei, XUE QingHong, CHEN XiaoChun. Establishment of Real-Time PCR Method for Detection of Extraneous Marek’s Disease Virus[J].Scientia Agricultura Sinica, 2023, 56(20): 4125-4136.

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Figures/Tables 9

Table 1

Sample information"

样品类型 Sample type	名称 Name	来源 Source
尿囊液生产毒 Viruses produced in allantoic fluid	鸡新城疫活疫苗 Newcastle Disease Vaccine, Live	某生产厂家A Manufacturer A
	鸡传染性支气管炎活疫苗 Avian Infectious Bronchitis Vaccine, Live	某生产厂家C Manufacturer C
	禽流感病毒 Avian Influenza Virus	某生产厂家D Manufacturer D
细胞生产毒 Viruses produced in cells	鸡传染性法氏囊病活疫苗 Infectious Bursal Disease Vaccine, Live	某生产厂家B Manufacturer B
	鸡痘活疫苗 Avian Pox Vaccine, Live	某生产厂家B Manufacturer B
	禽呼肠孤病毒 Avian Reovirus	中国兽医药品监察所实验室 Laboratory of China Institute of Veterinary Drug Control
	禽脑脊髓炎病毒 Avian Encephalomyelitis Virus	中国兽医药品监察所实验室 Laboratory of China Institute of Veterinary Drug Control
	禽腺病毒（I、III群） Avian Adenovirus（Group I、III）	中国兽医药品监察所实验室 Laboratory of China Institute of Veterinary Drug Control
	禽白血病病毒 Avian Leukosis Virus	中国兽医药品监察所实验室 Laboratory of China Institute of Veterinary Drug Control
	禽网内组织增生症病毒 Avian Reticuloendotheliosis Virus	中国兽医药品监察所实验室 Laboratory of China Institute of Veterinary Drug Control
组织（胚体/尿囊膜）生产毒 Viruses produced in tissues (embryonic body/allantoic membrane)	鸭瘟活疫苗 Duck Plague Vaccine, Live	某生产厂家F Manufacturer F
	鸡传染性喉气管炎活疫苗 Fowl Pox-Laryngotracheitis Vaccine, Live	某生产厂家E Manufacturer E
MDV 1毒株 MDV 1 strains	CVI988株 Strain CVI988	某生产厂家A Manufacturer A
	814株 Strain 814	某生产厂家G Manufacturer G
	SC 9-1株 Strain SC 9-1	某生产厂家H Manufacturer H
	Md5株 Strain Md5	中国兽医药品监察所实验室 Laboratory of China Institute of Veterinary Drug Control
MDV 3毒株 MDV 3 strains	FC126株 Strain FC126	中国兽医药品监察所实验室 Laboratory of China Institute of Veterinary Drug Control

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引物和探针 Primer and probes	序列 Sequence (5′-3′)
MDV 1-UL19-F	ATTCCTGGATGCAAACCCGT
MDV 1-UL19-R	TATTCCACGTTCCGTCAGCC
MDV 1-UL19-P	HEX-AGCCTGCCATCAGCGCGTAT-BHQ1
MDV 3-UL19-F	GCGATCACTGTCGCCTTCTA
MDV 3-UL19-R	AGTAACGGCTGTGGTCATGG
MDV 3-UL19-P	FAM-TGTAACGAGCGACGTCGTCCA-BHQ1

浓度（拷贝/μL） Content (copies/μL)	Ct值 Ct value			均值 X	标准差 SD	变异系数 CV (%)
浓度（拷贝/μL） Content (copies/μL)	1	2	3	均值 X	标准差 SD	变异系数 CV (%)
3.28×10⁷	14.41	14.16	14.33	14.30	0.13	0.91%
3.28×10⁶	16.23	16.22	16.25	16.23	0.01	0.09%
3.28×10⁵	19.66	19.48	19.69	19.61	0.11	0.57%
3.28×10⁴	24.17	24.14	24.16	24.16	0.01	0.05%
3.28×10³	27.83	27.76	27.91	27.83	0.07	0.27%
3.28×10²	30.96	31.11	31.09	31.05	0.08	0.27%
3.28×10¹	32.69	32.84	32.96	32.83	0.13	0.41%

浓度（拷贝/μL） Content (copies/μL)	Ct值 Ct value			均值 X	标准差 SD	变异系数 CV (%)
浓度（拷贝/μL） Content (copies/μL)	1	2	3	均值 X	标准差 SD	变异系数 CV (%)
1.00×10⁸	12.50	12.18	12.20	12.29	0.18	1.48%
1.00×10⁷	15.86	15.60	15.63	15.70	0.14	0.90%
1.00×10⁶	19.27	19.12	19.08	19.16	0.10	0.50%
1.00×10⁵	23.10	23.14	23.12	23.12	0.02	0.08%
1.00×10⁴	26.84	26.18	26.51	26.51	0.33	1.25%
1.00×10³	30.88	30.41	30.31	30.53	0.30	0.99%
1.00×10²	33.42	33.26	33.03	33.23	0.20	0.59%

Establishment of Real-Time PCR Method for Detection of Extraneous Marek’s Disease Virus

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