實驗小鼠致病性螺旋桿菌 多重引子聚合酶鏈鎖反應之建立

Abstract

螺旋桿菌屬（Helicobacter spp.）是實驗小鼠最常見的病原之一。多種囓齒動物致病性螺旋桿菌，包括H. hepaticus、H. bilis 和H. typhlonius，會影響免疫反應、有致癌性，並且可以改變生殖相關數據。目前，螺旋桿菌屬的PCR檢測已經應用在例行性小鼠健康監測。然而，目前臺灣尚無針對致病性的螺旋桿菌有PCR檢測方法。在本研究中，我們針對四種致病性螺旋桿菌，包含H. hepaticus、H. bilis、H. typhlonius及H. apodemus，以及大小鼠的管家基因，建立了一個多重引子聚合酶鏈鎖反應（Helicobacters/Actin multiplex PCR assay）。此Helicobacters/Actin多重引子聚合酶鏈鎖反應可特異性的增幅出四種螺旋桿菌的目標基因，並且能針對所有目標的螺旋桿菌之偵測極限達到50 copies 的高敏感性。為了調查這四種螺旋桿菌在台灣實驗小鼠的感染情況，本研究採用此Helicobacters/Actin多重引子聚合酶鏈鎖反應，針對來自10個不同研究機構119隻小鼠的糞便樣本進行感染情況的調查。此結果顯示，在10個不同的研究機構中有7個單位有這些致病性螺旋桿菌的感染，其感染率分別為： H. hepaticus（23.53%）、H. bilis（0%）、H. typhlonius（17.65%）和 H. apodemus（12.61%）。其中，含有多種螺旋桿菌共同感染情況分別為H. hepaticus/H. typhlonius（感染率為5.88%）和H. hepaticus/H. typhlonius/H. apodemus （感染率為8.40%）。並且，此多重引子聚合酶鏈鎖反應具有高敏感性（100%），高特異性（98.9% - 100%），高準確性（99.16% - 100%）。本研究開發的Helicobacters/Actin多重引子聚合酶鏈鎖反應在不需要犧牲動物及干擾動物的前提下，即可同時由實驗小鼠的糞便中，同時偵測四種致病性螺旋桿菌的感染

Helicobacter spp. is one of the most prevalent pathogens in laboratory mouse colonies. Several rodent helicobacters, including Helicobacter hepaticus, H. bilis, and H. typhlonius can modulate immunological responses, carcinogenesis, and reproduction related data. Currently, helicobacter generic PCR assays have been well applied in routine health monitoring of laboratory mice. However, species-specific PCR assays for pathogenic helicobacters are still not available in Taiwan. In this study, we developed a species-specific Helicobacters/Actin multiplex PCR assay to simultaneously detect and differentiate four helicobacter species (H. hepaticus, H. bilis, H. typhlonius, and H. apodemus) and a housekeeping gene in feces. The multiplex PCR assay could detect an equivalent infection of four helicobacter species, with a detection limit as few as 50 copies. To survey the infection status of these four helicobacter species, a total of 119 mouse fecal samples collected from 10 different research facilities were screened by this multiplex PCR assay. The prevalence surveillance result revealed that 7 out of 10 different research facilities were positive for helicobacters and the infection rates were as followed: H. hepaticus (23.53%), H. bilis (0%), H. typhlonius (17.65%), and H. apodemus (12.61%). In addition, multiple co-infection rates of helicobacters species were 5.88% (H. hepaticus/H. typhlonius) and 8.40% (H. hepaticus/H. typhlonius/H. apodemus). This multiplex PCR assay has a high sensitivity (100%), specificity (98.9% - 100%), and accuracy (99.16% - 100%). The Helicobacters/Actin multiplex PCR assay developed in this study would be a powerful tool to screen pathogenic helicobacter infections in laboratory mouse colonies without animal sacrifice.