彎曲桿菌及空腸彎曲桿菌之分子鑑定及PCR與生物晶片發展

Abstract

Campylobacter spp. is one of the major food-borne pathogenic species and in recent year, the incidence increased rapidly. Campylobacter spp. cause serious infections in humans, including diarrhea, abdominal pain, bloody diarrhea and septicemia or the more serious syndrome, Guillain-Barr. Campylobacter spp. are not paid with attention by the breeders and verterinarians because it rarely infected the grallatores. However, it could contaminate the carcass during the meat processing. Thus, Campylobacter spp. play an important role on public health. Several commercial kits for identification of Campylobacter spp. have been developed. These methods include traditional biochemical identification, DNA probe, ELISA and Latex Test Kit. Molecular methods, such as PCR, PCR enzyme-linked immunosorbent assay and real-time PCR assay have also successfully been used for Campylobacter spp. identification. In this study, we used PCR primers MD16S1/MD16S2, Camp F/Camp R, OT1559/18-1, C-1/C-4 and CC18F/CC519R for the preliminary screening of C. jejuni isolates in our lab. Then we developed primers, Hsp54s/Hsp211a from hsl V gene of C. jejuni which permits the specific PCR detection of C. jejuni. Using Hsp54s/Hsp211a, only the DNA from C. jejuni generated the expected PCR product equal to 157 bp. Bacteria strains other then C. jejuni including strains of other Campylobacter species, would not give any false positive result. When Hsp54s/Hsp211a was used for monitoring of C. jejuni in chicken samples, a detection limit of 104 CFU/ml could be obtained in the conventional PCR and a detection limit 100 CFU/ml could be obtained for real-time PCR. After 18H enrichment step prior to the PCR, the detection sensitivity could reach to 100 CFU/ml of the food homogeneous liquid for conventional PCR. By alignment the sequences of PCR products amplified with the primers we developed, the amplified PCR sequence is unique to C. jejuni. This result indicates that these sequences have potential to be used as probes on microarray. When large numbers of isolates are assayed, microarray is powerful tool for detected such a mass genes and offer a platform of rapid and complete detecion. In this report, we also developed a microarray for detection the gene hsl V of C. jejuni. For such array, oligonucleotide probe derived from hsl V gene, probe - super probe was spotted on the Nylon membrane. After hybridization of the biotin-labeled PCR product amplified from Hsp54s/Hsp211a primers, the results showed that all the tested isolates of C. jejuni could be correctly identified.

彎曲桿菌是近來在食物傳播病出現的病原菌，其發生率也迅速增加。除了會引起人類下痢、腹痛、血痢甚至是敗血症或是更嚴重的自體免疫疾病─格林貝利症候群(Guillain-Barre syndrome)；但對禽類致病性極低、不受畜主與獸醫重視，卻能在屠宰加工過程中汙染禽類屠體，因此在公共衛生上扮演極具重要的角色。 目前已有許多用於鑑定彎曲桿菌的商業套組，包含了傳統的生化鑑定、核酸探針、ELISA及膠凝集試驗；而分子檢測方法，例如PCR、酵素連結免疫螢光PCR或是即時定量PCR也已經成功應用在彎曲桿菌的鑑定上。本研究先使用學者所發表針對彎曲桿菌進行特異性檢測之PCR引子組MD16S1/MD16S2、Camp F/Camp R、OT1559/18-1、c-1/c-4及CC18-F/CC519-R進行實驗室空腸彎曲桿菌之初步鑑定。 本研究針對空腸彎曲桿菌熱休克蛋白基因設計一組具特異性引子組Hsp54s/Hsp211a並進行特異性檢測。結果顯示，除了空腸彎曲桿菌會產生157 bp之預期目標產物外，其他菌株(包含非空腸彎曲桿菌的彎曲桿菌屬菌株)，皆不會產生偽陽性之干擾。當引子組Hsp54s/Hsp211a應用於檢測雞肉樣品中空腸彎曲桿菌時， 傳統PCR 靈敏度可以達到104CFU/ml，而在增殖18小時後，其傳統PCR靈敏度可以達到原始菌數為100CFU/ml；real-time PCR靈敏度則可以達到100CFU/ml。將自行設計之特異性PCR引子組所增幅出來之PCR產物進行序列比對，結果顯示整段序列均具有特異性、具有發展成為生物晶片探針之潛力。 在面對大量樣本時，高密度的基因晶片技術可以快速地解決病原菌鑑定上費時費力的問題，提供一個可以快速且整體性的實驗平台。因此本研究利用空腸彎曲桿菌的熱休克蛋白基因，發展檢測空腸彎曲桿菌用之雛型基因晶片，發現此基因晶片確實能有效鑑別空腸彎曲桿菌。