非流感呼吸道病毒之多重即時聚合酶鏈鎖反應

Abstract

呼吸道感染為全球前十大死因之一，而常見的呼吸道感染以病毒所引發的類流感(Influenza-like illness，ILI)為主；其病原體除了季節性A型流感病毒(Influenza A viruses)、季節性B型流感病毒(Influenza B viruses)、副流感病毒(Parainfluenza viruses，PIV)、呼吸道融合病毒(Respiratory syncytial virus，RSV)、腺病毒(Adenovirus，ADV)，亦包含新興病毒，如:人類間質肺炎病毒(Human Metapneumovirus，hMPV)及人類博卡病毒(Human Bocavirus，HBoV)。統計台大醫院2018年1 月至2019年4月呼吸道檢體之各項檢驗結果，包含流感快篩、流感病毒PCR及病毒培養，發現大部分檢體結果呈現陰性，無法確認其致病因，因此須仰賴分生檢驗技術進行更精確之檢測。本研究之目的即為發展可同步檢驗多種呼吸道病毒之即時分子診斷平台。本篇研究目的為利用TaqMan® 探針發展具專一性之即時多重螢光定量平台，可同步檢驗八種呼吸道病毒：人類博卡病毒、人類間質肺炎病毒、A型與B型呼吸道融合病毒、第一型至第三型副流感病毒及腺病毒。建立平台之首要步驟為針對各病毒之保守序列設計專一性引子及探針。首先至NCBI基因資料庫中收集已發表之呼吸道病毒序列，所選取序列需涵蓋不同年份、區域與基因型別，再經由序列分析軟體MEGA6進行序列分析找出保守序列後設計具專一性的引子與探針。接著建立實驗所需的對照組，將實驗室已保存之呼吸道病毒及臨床呼吸道檢體，利用前述設計之引子夾出病毒片段，嵌入pCRII-TOPO載體製備成病毒標準品，而後將其定序後所得之序列以Blast (Basic local alignment search tool)確認病毒序列、繪製親緣關係樹 (Phylogenetic Tree)，以確認標準品之病毒基因型。同時，為確認各病毒引子的專一性、排除引子對於它種病毒具交互反應(Cross-reactivity)，將不同的引子與病毒標準品配對後進行PCR，其結果證明本研究之引子皆具有高度專一性、未觀察到假性結合(False binding)之現象。接著利用SYBR green與TaqMan® 探針進行Simplex Real-time PCR實驗條件測試，利用病毒標準品設立標準曲線後，確認引子與探針之實驗條件，並測試平台之偵測範圍，其實驗結果顯示除了人類博卡病毒及第一、二型副流感病毒之偵測範圍為104至108 copies/5 μL，其餘皆從103至108 copies/5 μL。此外，同時利用已知病毒核酸及臨床檢體進行測試，證明Simplex Real-time PCR偵測結果與已知結果相符。在確認上述條件皆完備後即進行Multiplex Real-time PCR的測試，其結果顯示Multiplex Real-time PCR偵測範圍皆與Simplex Real-time PCR一致；在多重即時聚合酶鏈鎖反應中也未觀察到同組內三種病毒間之交互反應，顯示本研究多重即時聚合酶鏈鎖反應具有高度專一性。最後，我們收集2019年1月至2019年4月間，114支台大病毒室未確定感染源之肺炎患者的臨床檢體，經由本平台之多重聚合酶鏈鎖反應檢驗後，有32支檢體為陽性，分別為: A型呼吸道融合病毒3支，B型呼吸道融合病毒1支，人類間質肺炎病毒4支，第一型副流感病毒2支，第二型副流感病毒3支，第三型副流感病毒5支，人類博卡病毒2支，腺病毒11支，及第二型副流感病毒與腺病毒共同感染1支，檢驗陽性率為28%，顯示本平台之多重聚合酶鏈鎖反應具有高度專一性及敏感度，可應用於臨床檢驗及疫情防治。

Respiratory infection was one of the top 10 causes that led to deaths worldwide, and the Influenza-like illness (ILI) which arose from virus infection was the major cause of respiratory infection. The ILI pathogens include influenza A virus, influenza B virus, parainfluenza virus (PIV), respiratory syncytial virus (RSV), adenovirus (ADV) and some emerging viruses, such as human metapneumovirus (hMPV) and human bocavirus (HBoV). After analyzing the diagnosis results of respiratory specimens from National Taiwan University Hospital between January 2018 and April 2019, including influenza rapid screening, influenza virus PCR and virus culture, we found that most of the diagnosis was negative and could not confirm the pathogens responsible for respiratory infections. Therefore, it is necessary to apply the molecular technology for more accurate detection of virus pathogen. The study is aimed to develop the multiplex real-time PCR to detect eight common respiratory viruses simultaneously, including influenza A viruses, influenza B viruses, PIV, RSV, ADV and 2 emerging viruses, HMPV and HBoV. The first step was to collect the sequences of respiratory viruses from NCBI deposited at different years, regions and genotypes, then to design specific primers and probes for the conserved sequences for each virus. Next, we constructed the control plasmids by cloning the respective virus PCR products into the pCRII-TOPO vectors. The Blast (Basic local alignment search tool) and phylogenetic analysis was then conducted to confirm the viral sequences and genotypes. The specificity of each virus primers was confirmed by PCR. The SYBR® Green and TaqMan® probes were used in the simplex real-time PCR to determine the experimental conditions and the detection ranges for each virus. The detection range for BoV, PIV-1 and PIV-2 was 104 to 108 copies/5μL, the others were 103 to 108 copies/5μL. The performance of the PCR was confirmed using clinical samples with confirmed virus identity. After establishment of simplex real-time PCR for each virus, 3 sets of multiplex real-time PCR were designed. The respective viral plasmids were used to test the experimental conditions for each set of multiplex real-time PCR. The Ct values of standard reagents were compared between simplex real-time PCR and multiplex real-time PCR. The detection range in multiplex real-time PCR was the same as the detection range in the simplex real-time PCR. The interaction between the 3 viruses in the same group was not observed, indicating that multiplex real-time PCR in this study is highly specific. Then, the respiratory sepcimens or cultured viruses were used to compare the Ct values of simplex and multiplex real-time PCR. The results showed that the Ct values of multiplex real-time PCR were similar to the Ct values of the simplex real-time PCR. Finally, we used the platform to examine the potential pathogens of 114 respiratory specimens from pneumonia cases which were collected from January 2019 to April 2019 in National Taiwan University Hospital and the pathogens were not defined. Among the 114 original specimens, 32 (28%) were found positive for at least one virus. RSV-A, RSV-B, HMPV, PIV-1, PIV-2, PIV-3, BoV and ADV were detected in 3, 1 , 4 , 2, 3 , 5, 2 and 11 of the specimens, respectively. Dual infection accounted for 3.13% (1/32) of all positive specimens. The study results indicate that the designed platform could facilitate the identification of respiratory pathogens, which could not be identified using the conventional virus culture.