本文選題：空腸彎曲桿菌 + 產(chǎn)氣莢膜梭菌　； 參考：《安徽農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：隨著人類(lèi)生活水平的不斷提高,禽類(lèi)產(chǎn)品在人類(lèi)食物中所占的比重日益加大,然而空腸彎曲桿菌和產(chǎn)氣莢膜梭菌等人畜共患病原菌可污染禽類(lèi)產(chǎn)品,引起人類(lèi)細(xì)菌性疾病,且其發(fā)生呈逐漸增長(zhǎng)趨勢(shì)。空腸彎曲桿菌和產(chǎn)氣莢膜梭菌不僅嚴(yán)重影響?zhàn)B禽業(yè)的發(fā)展,造成經(jīng)濟(jì)損失,同時(shí)也是造成人類(lèi)胃腸炎等疾病的主要病原菌,嚴(yán)重威脅人類(lèi)健康。目前,對(duì)食品中食源性致病菌定量檢測(cè)的方法主要為活菌平板計(jì)數(shù),耗時(shí)費(fèi)力,很難計(jì)算準(zhǔn)確的數(shù)量;MPN法(最大可能數(shù))需要進(jìn)行生化鑒定,同樣操作繁瑣,耗時(shí)費(fèi)力;而(Real-time)PCR檢測(cè)則無(wú)法判斷樣品中的細(xì)菌是否為活菌。因此,研究旨在建立針對(duì)空腸彎曲桿菌和產(chǎn)氣莢膜梭菌的MPN-PCR檢測(cè)方法,并初步應(yīng)用于雞肉制品中這兩種病原菌的快速定量檢測(cè)。首先,通過(guò)查閱相關(guān)文獻(xiàn),根據(jù)空腸彎曲桿菌特有的馬尿酸酶hipO基因和產(chǎn)氣莢膜梭菌各基因型均編碼的cpa基因設(shè)計(jì)特異性引物,將PCR技術(shù)與MPN法相結(jié)合,建立了MPN-PCR快速定量檢測(cè)方法,用于定量檢測(cè)雞肉制品中的空腸彎曲桿菌和產(chǎn)氣莢膜梭菌。MPN-PCR方法的整個(gè)檢測(cè)過(guò)程包括:樣品處理、增菌培養(yǎng)、模板制備、PCR擴(kuò)增,實(shí)現(xiàn)了對(duì)食品中病原菌的快速定量檢測(cè)。用PCR技術(shù)替代了生化試驗(yàn),不僅可以進(jìn)行定量檢測(cè),還可以避免了由于細(xì)菌進(jìn)入VBNC(活的但非可培養(yǎng))狀態(tài)所造成的漏檢,并且檢測(cè)時(shí)間至少縮短了兩天以上。其次,應(yīng)用建立的MPN-PCR定量檢測(cè)方法,對(duì)采集的186份雞肉樣品進(jìn)行實(shí)際應(yīng)用,其中鮮雞肉66份,凍雞肉120份。結(jié)果:空腸彎曲桿菌的陽(yáng)性檢出率為17.2%,鮮雞肉的陽(yáng)性檢出率為28.79%,凍雞肉的陽(yáng)性檢出率為10.83%,含菌量范圍在3.6-92 MPN/g;產(chǎn)氣莢膜梭菌的陽(yáng)性檢出率為15.59%,鮮雞肉的陽(yáng)性檢出率為13.64%,凍雞肉的陽(yáng)性檢出率為16.67%,含菌量范圍在3.6-360 MPN/g。綜上所述,試驗(yàn)建立的空腸彎曲桿菌和產(chǎn)氣莢膜梭菌MPN-PCR檢測(cè)方法,具有特異性強(qiáng)、敏感性高、相對(duì)快速、高效等特點(diǎn),能為養(yǎng)禽業(yè)中疾病的防控和禽肉類(lèi)加工銷(xiāo)售過(guò)程的監(jiān)管提供有效的工具,對(duì)食品公共衛(wèi)生安全領(lǐng)域中食源性病原菌的檢測(cè)提供一定的幫助。
[Abstract]:With the improvement of human living standards, poultry products account for an increasing proportion of human food. However, zoonotic pathogens such as Campylobacter jejuni and Clostridium perfringens can pollute poultry products and cause human bacterial diseases. And its occurrence shows a gradual increase trend. Campylobacter jejuni and Clostridium perfringens not only seriously affect the development of poultry industry, resulting in economic losses, but also are the main pathogens causing gastroenteritis and other diseases, which seriously threaten human health. At present, the main method for quantitative detection of foodborne pathogenic bacteria in food is the counting of live bacteria, which is time-consuming and laborious, so it is difficult to calculate the exact quantity of MPN (maximum possible number), which requires biochemical identification, and the same operation is cumbersome and time-consuming. Real-time PCR could not determine whether the bacteria in the sample were living bacteria. Therefore, the aim of this study was to establish a MPN-PCR method for the detection of Campylobacter jejuni and Clostridium perfringens, and to apply them to the rapid quantitative detection of these two pathogens in chicken products. Firstly, specific primers were designed according to the specific cpa gene encoding the hipO gene of Campylobacter jejuni and the cpa gene of Clostridium perfringens. The PCR technique was combined with the MPN method. A rapid and quantitative MPN-PCR method was established for quantitative detection of Campylobacter jejuni and Clostridium perfringens. The rapid quantitative detection of pathogenic bacteria in food was realized. The biochemical test can be replaced by PCR technique, which can not only detect quantitatively, but also avoid the missed detection caused by bacteria entering the state of VBNC (living but not culturable), and the detection time is shortened by more than two days. Secondly, 66 fresh chicken samples and 120 frozen chicken samples were tested by the established MPN-PCR quantitative detection method. Results: the positive rates of Campylobacter jejuni, fresh chicken and frozen chicken were 17.2, 28.79, 10.83, 3.6-92 MPN / g, 15.59 and 10.83, respectively. The positive rate of frozen chicken was 16.67 and the amount of bacteria was in the range of 3.6-360 MPN / g. In conclusion, the MPN-PCR method for detection of Campylobacter jejuni and Clostridium perfringens has the characteristics of high specificity, high sensitivity, relatively fast and high efficiency. It can provide effective tools for the prevention and control of diseases in poultry industry and the supervision of poultry meat processing and marketing process. It can also provide some help for the detection of foodborne pathogens in the field of food public health and safety.
【學(xué)位授予單位】：安徽農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：S852.61

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1 韓梅;MPN-PCR法檢測(cè)雞肉中空腸彎曲桿菌和產(chǎn)氣莢膜梭菌[D];安徽農(nóng)業(yè)大學(xué);2015年

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本文編號(hào)：1869580