本文選題：多重PCR + 沙門氏菌�。� 參考：《福建農林大學》2013年碩士論文

【摘要】：當前社會,由食物源病原微生物引起的人類疾病和死亡病例越來越引起人們的關注。為了保障自身的健康,居民們對食物供給的安全性和食用性有了更多的需求。這些需求要求當前的食品檢測技術必須不斷發(fā)展進步,與時俱進。傳統(tǒng)微生物檢測方法雖然仍被使用,但是它耗時費力,并且檢測靈敏度也不高,并且需要受過專業(yè)培訓經驗豐富的檢測人員才能勝任。分子生物學的的發(fā)展極大地改變了食品檢測技術。免疫學技術、分子生物學技術、自動化以及信息化技術的正向推動作用已經被被大家看在眼里。我們應該勇于探索將不同的檢測技術聯(lián)合使用的可能性。最新的檢測技術正向著病原微生物定量檢測和同時檢測多個靶標微生物或毒素這個方向發(fā)展。 相比于傳統(tǒng)PCR,多重PCR含有更高的技術含量,把兩對以上的引物加入到同一個反應體系中,使它們針對各自的目的基因經行同時擴增,最終可以同時得到不同病原微生物的檢測結果,大大縮短了檢測時間,同時也節(jié)約了檢測成本。 本研究意在建立一種可用于同時檢測四種食源性腸道致病菌的多重PCR方法。此前,沒有針對弗氏檸檬酸桿菌的分子生物學檢測方法。首先依據沙門氏菌(Salmonella spp)的fimY基因,弗氏枸櫞酸桿菌(Citrobacter freundii)的ldh基因,奇異變形桿菌(Proteus mirabilis)的idsC基因和遲緩愛德華氏菌(Edwardsiella tarda)的fimA基因的保守序列設計了四對特異性引物,其PCR擴增產物大小依次為161bp、336bp、396bp、526bp。然后建立了針對四種致病菌的單重PCR檢測體系,并通過試驗確定了特異性和靈敏度。通過測序確定特異性極高,體系中檢測到沙門氏菌的靈敏度為39CFU；體系中檢測到弗氏檸檬酸桿菌的靈敏度為72CFU；體系中檢測到奇異變形桿菌的靈敏度為6CFU；體系中檢測到遲緩愛德華氏菌的靈敏度為7CFU。這為之后建立多重PCR檢測體系提供了很好的參考。 在四種細菌的單重PCR基礎上,根據對多重PCR反應的主要影響因素,如退火溫度,引物濃度,dNTP濃度和Mg2+濃度等,將這些因素展開試驗進行優(yōu)化,建立了四種細菌的多重PCR檢測體系。研究結果表明該多重PCR反應體系對4株病原菌能擴增出清晰特異的條帶。并且體系中沙門氏菌的檢測靈敏度為39CFU；弗氏檸檬酸桿菌的檢測靈敏度為7CFU；奇異變形桿菌的檢測靈敏度為57CFU；遲緩愛德華氏菌的檢測靈敏度為6CFU。 人工染菌樣品檢測結果顯示,通過預增菌后,樣品中目的菌濃度在10CFU/ml數量級時結果為陽性,說明本研究建立的多重PCR方法具有結果可靠、靈敏度高、方便快捷的優(yōu)點。本檢測體系十分適用于針對四種食源性腸道致病菌的檢測,在食品加工過程以及食品進出口環(huán)節(jié)都有很廣的應用空間。并且為研發(fā)快速檢測以上四種食源性腸道致病菌的試劑盒提供了重要技術保障。 同時,本研究還初步探索了利用基質輔助激光解析電離飛行時間質譜儀對四種細菌的檢測方法,通過與己建立的單重PCR方法比較,可以得知該方法具有特異性高、操作便捷的優(yōu)點。
[Abstract]:In current society, caused by a food source pathogenic microorganism in human diseases and deaths caused by more and more attention. In order to protect their own health, the residents of food safety and food have more demand. The demand of food inspection technology current must be continuous development progress, conventional methods of keeping pace with the times. Although it is still used, but it is time-consuming, and the detection sensitivity is not high, and the need for trained experienced inspectors to be competent. Molecular biology development has greatly changed the food inspection technology. Immunological techniques, molecular biology techniques, positive role in promoting automation and information technology have been for everyone to see in the eyes. We should have the courage to explore the possibility of the combined use of different detection techniques. The new detection technology is disease The quantitative detection of microbes and the simultaneous detection of multiple target microorganisms or toxins have developed in this direction.
Compared to the conventional PCR, multiplex PCR with higher technology content, to more than two pairs of primers were added to the same reaction system, making them for their target genes by simultaneous amplification can be obtained at the same time, the final detection results of different pathogenic microorganisms, shorten testing time, but also saves the cost of detection.
This study aimed to establish a kind of can be used for simultaneous detection of four foodborne pathogens of intestinal multiple PCR method. Previously, no molecular biological detection methods for c.freundii. Firstly on the basis of Salmonella (Salmonella spp) fimY gene, Citrobacter freundii (Citrobacter freundii) LDH gene, proteus coli (Proteus mirabilis) idsC gene and delayed Edward's bacteria (Edwardsiella tarda) conserved sequence of fimA gene was designed four pairs of specific primers, PCR amplification of 161bp, 336bp, 396bp, 526bp. and then set up for the four single PCR pathogen detection system, and determine the specific and through the test. To determine the sensitivity of high specificity by sequencing system, detect sensitivity of Salmonella 39CFU system; detected c.freundii sensitivity to 72CFU body; The sensitivity of the Proteus mirabilis detected in the system was 6CFU. The sensitivity of Edward's bacteria detected in the system was 7CFU., which provided a good reference for the establishment of multiple PCR detection system after that.
In a single PCR based on four kinds of bacteria, according to the main influencing factors of response to multiple PCR, such as annealing temperature, primer concentration, dNTP concentration and Mg2+ concentration, these factors test optimization, establish multiple PCR detection system of four kinds of bacteria. The results showed that the multiplex PCR reaction system of 4 strains of pathogenic bacteria can be amplified and specific. The detection sensitivity of Salmonella in the system is 39CFU; the detection sensitivity of c.freundii was 7CFU; Bacillus proteus detection sensitivity is 57CFU; Edwardsiella detection sensitivity is 6CFU.
Artificial infection sample test results showed that the pre enrichment, the purpose of bacteria concentration in the samples in the level of 10CFU/ml when the result is positive, that the multiplex PCR method established in this study is reliable, high sensitivity, convenient advantages. The detection system is very suitable for the detection of four food borne pathogens needle intestine. Have a very wide application space in the process of food processing and food import and export sectors. Provides important technical guarantee for the development of pathogens and kits for rapid detection of more than four species of food borne.
At the same time, this study also explored the detection method of four kinds of bacteria by matrix assisted laser desorption ionization time of flight mass spectrometry. Comparing with the established single PCR method, we can see that this method has the advantages of high specificity and convenient operation.

【學位授予單位】：福建農林大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R155.5

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