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水禽細小病毒SYBR Green I熒光定量PCR檢測方法的建立與應用

2024-07-04 21:17:19汪宏才商雨馬瑤曾哲張蓉蓉姚倫羅玲李麗溫國元羅青平
湖北農業科學 2024年6期

汪宏才 商雨 馬瑤 曾哲 張蓉蓉 姚倫 羅玲 李麗 溫國元 羅青平

摘要:為了建立水禽細小病毒(WPV)快速檢測方法,根據序列比對結果在水禽細小病毒NS基因SF3保守區域內設計特異性引物,建立SYBR Green I熒光定量PCR通用檢測方法。該方法的擴增效率(E)為90.0%,相關系數(R2)=0.99,標準曲線方程為y=-3.607x+38.77;除WPV出現S形擴增曲線外,新城疫病毒(NDV)、H9亞型禽流感病毒(H9 AIV)、鴨坦布蘇病毒(DTMUV)、鴨肝炎病毒(DHAV)、鴨腸炎病毒(DEV)、鴨呼腸孤病毒(DRV)樣品均未出現S形陽性擴增曲線;批內變異系數(CV)為0.15%~0.23%,批間變異系數為0.09%~0.28%。結果表明,SYBR Green I熒光定量PCR檢測方法重復性好、靈敏度高和特異性強。臨床樣品檢測結果表明,SYBR Green I熒光定量PCR與普通PCR的符合率達98.4%,靈敏度是普通PCR的1 000倍。SYBR Green I熒光定量PCR檢測方法不僅能定性檢測WPV,還可以進行定量檢測,可用于種鴨場、種鵝場的WPV凈化檢測,也可用于WPV臨床大量樣品的快速檢測。

關鍵詞:水禽細小病毒;檢測方法;SYBR Green I;熒光定量PCR

中圖分類號:S855.3? ? ? ? ?文獻標識碼:A

文章編號:0439-8114(2024)06-0218-08

DOI:10.14088/j.cnki.issn0439-8114.2024.06.036 開放科學(資源服務)標識碼(OSID):

Establishment and application of a SYBR Green I fluorescence quantitative PCR detection method for waterfowl parvoviruses

WANG Hong-cai1,2,SHANG Yu1,2,MA Yao1a,ZENG Zhe1,2,ZHANG Rong-rong1,2,YAO Lun1,2,

LUO Ling1,2,Li Li1a,WEN Guo-yuan1,2,LUO Qing-ping1,2

(1a.Institute of Animal Husbandry and Veterinary; 1b. Key Laboratory of Animal Bacterial Disease Prevention and Control Formulations of the Ministry of Agriculture and Rural Affairs; 1c.Hubei Key Laboratory of Pathogenic Microbiology of Livestock and Poultry, Hubei Academy of Agricultural Sciences ,Wuhan 430064, China; 2. Hubei Hongshan Laboratory,Wuhan 430070, China)

Abstract: In order to establish a rapid detection method for waterfowl parvoviruses (WPV), specific primers were designed within the conserved SF3 region of the NS gene of waterfowl parvoviruses based on sequence alignment results, and a SYBR Green I fluorescence quantitative PCR universal detection method was established. The amplification efficiency (E) of this method was 90.0%, the correlation coefficient (R2) was 0.99, and the standard curve equation was y=-3.607x+38.77;except for WPV with an S-shaped amplification curve, the newcastle disease virus (NDV), H9 subtype avian influenza virus (H9 AIV), duck tembusu virus (DTMUV), duck hepatitis A virus (DHAV), duck enteritis virus (DEV), and duck reovirus (DRV) samples did not show an S-shaped positive amplification curve;the coefficient of variation (CV) within a batch was 0.15% to 0.23%, and the coefficient of variation between batches was 0.09% to 0.28%. The results indicated that the SYBR Green I fluorescence quantitative PCR detection method had good repeatability, high sensitivity, and strong specificity. The clinical sample testing results showed that the coincidence rate between SYBR Green I fluorescence quantitative PCR and conventional PCR was 98.4%, and the sensitivity was 1 000 times higher than that of conventional PCR. The SYBR Green I fluorescence quantitative PCR detection method could not only qualitatively detect WPV, but also quantitatively detect it. It could be used for WPV purification detection in duck and goose breeding farms, as well as for rapid detection of WPV in large clinical samples.

Key words: waterfowl parvoviruses; detection method; SYBR Green I; fluorescence quantitative PCR

水禽細小病毒(Waterfowl parvoviruses,WPV)屬于雁形目依賴細小病毒1型(Anseriform dependoparvovirus 1)[1],包括鵝細小病毒(Goose parvovirus, GPV)、番鴨細小病毒(Muscovy duck parvovirus,MDPV)和鴨細小病毒(Duck parvovirus,DPV),三者在病毒形態、培養特性、理化性質及基因組結構等方面具有相似性,但致病性和抗原性卻存在很大差異[2]。GPV主要引起雛鵝和番鴨發病,MDPV僅引起番鴨發病,而對鵝、鴨等不致病,DPV是GPV變異株,主要引起鴨發生短喙侏儒綜合征(Short beak and dwarfism, SBDS)。WPV具有二十面體結構,無包膜,基因組為線性單鏈DNA,大小為4~6 kb[3]。基因組包含2個主要的開放閱讀框,編碼非結構蛋白(Non-structural protein, NS)和結構蛋白(Viral structural protein, VP)。最大的NS蛋白是一種多結構域蛋白,含有高度保守的解旋酶超家族3(Superfamily 3, SF3)結構域,具有解旋酶和ATP酶活性[4]。……

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