以巢式RT-PCR偵測石蠟包埋組織中貓冠狀病毒作為輔助病理診斷之方法

Molecular Detection of Feline Coronaviruses in Formalin-Fixed and Paraffin-Embedded Tissue (FFPE) by nested RT-PCRs: a Diagnosis-Aiding Approach

郭 軒(Shanny Hsuan Kuo)

指導教授 ： 張惠雯

共同指導教授 ： 龐 飛 ； 鄭謙仁(Chian-Ren Jeng)

國立臺灣大學/獸醫專業學院/分子暨比較病理生物學研究所/碩士(2017年)

https://doi.org/10.6342/NTU201702851

摘要

貓傳染性腹膜炎（Feline infectious peritonitis, FIP）是由貓冠狀病毒（feline coronavirus, FCoV）所引起的貓隻致死性疾病。其非特異性的臨床症狀使此疾病生前診斷仍然非常困難。應用組織病理學檢查合併免疫組織化學染色（Immunohistochemistry, IHC）是目前公認診斷FIP的黃金標準。然而IHC的診斷敏感度卻會大大受限於組織中含有病毒抗原的細胞量的多寡。小樣本量採樣以及模棱兩可的IHC染色訊號分佈等局限性使得常用於組織病理學檢查的福馬林固定和石蠟包埋組織（FFPE）之生檢組織檢查成為獸醫病理學家診斷FIP最具挑戰性的樣材之一。先前文獻已證明，常規針對3′非轉譯區（3' untranslated region, 3'UTR）進行增幅的聚合酶連鎖反應（Polymerase chain reaction, PCR）對於新鮮組織中FIP的診斷是非特異性的。此外，近期有兩個在突起蛋白基因（Spike protein gene, S gene）被發現的突變，分別是S基因中位於1058處的甲氧氨基（M）至亮氨酸（L）氨基酸突變（M1058L）與絲氨酸（S）至丙氨酸（A）氨基酸突變（S1060A），兩者一起可以在血清型I貓冠狀病毒感染病例中，區分大於 95％貓傳染性腹膜炎病毒（Feline infectious peritonitis virus, FIPV）與貓腸道型冠狀病毒（Feline enteric coronavirus, FECV），而該發現具有潛在的診斷價值。本研究的目的是比較常規針對3′非轉譯區（Consensus nRT-PCR targeting 3’UTR）與針對S基因兩個突變（nRT-PCR targeting the two mutations in S gene）的巢式反轉錄聚合酶鏈反應（nested reverse transcription polymerase chain reaction, nRT-PCR）兩診斷技術的差異以提升由FFPE組織中進行FIP的診斷。透過針對貓隻GAPDH的持家基因的反轉錄聚合酶鏈反應（RT-PCR）評估FFPE組織中RNA的質量後，本研究之最終研究群體包括38個組織病理學與免疫組織化學證實的FIP貓隻和22隻非FIP貓隻。本實驗利用這些臨床與組織病理學下懷疑FIP案例的FFPE組織檢體作為RNA的來源進行巢式反轉錄聚合酶鏈反應（nRT-PCR）。本實驗使用常規針對3′非轉譯區的nRT-PCR成功在31/38（82％）FFPE FIP樣品中提取了RNA與擴增貓冠狀病毒核酸，而利用S基因特異性nRT-PCR可檢測到17/38（42％）陽性病例，此外對血清型1病例進行定序與後續氨基酸比對後發現17例中有16例在S基因中出現M1058L和S1060A兩個突變之其中一個。兩種方法均未於非FIP貓的FFPF組織檢出陽性結果。本研究發現，結合組織病理學和IHC染色結果，常規的nRT-PCR和S特異性nRT-PCR皆能夠成功檢測來自FFPE樣本的貓冠狀病毒RNA，且此方法可成功應用於IHC信號不明確而可能被誤解為陰性的病例中以提升FIP診斷效率，這兩種方法都可作為FIP診斷和由FFPE組織進行FIP回顧性研究的有用工具。

關鍵字

貓傳染性腹膜炎 ； 貓冠狀病毒 ； 福馬林固定和石蠟包埋組織 ； 巢式反轉錄聚合酶鏈反應 ； S基因突變

並列摘要

Feline infectious peritonitis (FIP), caused by feline coronavirus (FCoV), is a lethal disease in cats. The clinical signs are non-specific and antemortem diagnosis remains challenging and frustrating. Appling histopathology combined with immunohistochemical (IHC) staining is considered as the gold standard for FIP diagnosis. However, the sensitivity of the IHC method depends much on the numbers of intralesional antigen-bearing cells. Due to the limitations of small sampling sizes as well as the equivocal IHC staining pattern in some specimens, formalin-fixed and paraffin-embedded tissue (FFPE) biopsies frequently submitted for histopathological examination for FIP are the most challenging specimens for pathologists. It has been demonstrated that the consensus PCR targeting 3’UTR alone is non-specific for diagnosis of FIP in fresh tissues. Moreover, two recently described mutations, the substitution of methionine (M) to leucine (L) amino acid mutation at position 1058 (M1058L) and the substitution of serine (S) to alanine (A) amino acid mutation at position 1060 (S1060A) in spike (S) gene, which together can distinguish feline infectious peritonitis virus (FIPV) from feline enteric coronavirus (FECV) in >95% of serotype I FCoV-infected cases in freshly-collected specimens, have suggested a potential diagnostic value. The aim of this study was to compare the uses of a consensus nested RT-PCR (nRT-PCR) targeting 3’UTR and a nRT-PCR targeting the two mutations in S gene in aiding the diagnosis of FIP in FFPE tissues. After evaluation of the RNA quality in FFPE tissues by a RT-PCR targeting the housekeeping gene of feline GAPDH, a total of 38 histopathologically and immunohistochemically confirmed FIP cases and 22 non-FIP cases were used as the source of RNA and examined nRT-PCRs. We have successfully extracted RNA and amplified FCoV genes in 31/38 (82%) FIP cases using consensus nRT-PCR, whereas 17/38 (42%) FIP cases were detected using the S-specific nRT-PCR. Following subsequent sequencing, 16 out of 17 serotype 1 cases had one of the two mutations (M1058L and S1060A) in the S gene. None of the FFPF tissues from these non-FIP cats were positive by both methods. We have demonstrated that in combined with histopathology and IHC staining, both consensus nRT-PCR and S-specific nRT-PCR were capable of detecting viral RNA from FFPE samples where IHC signals were equivocal and possibly misinterpreted as negativity. Both methods serve as a useful tool in supporting FIP diagnosis and for the retrospective study of FIP in archival FFPE tissues.

並列關鍵字

Feline infectious peritonitis (FIP) ； feline coronavirus ； formalin-fixed and paraffin-embedded tissue ； RT-PCR ； spike mutation

參考文獻

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