【作者】 常玉华；

【导师】 仇农学；

【作者基本信息】 陕西师范大学 ， 食品科学， 2003， 硕士

【摘要】 耐热菌，即酸土环脂芽孢杆菌（Alicyclobacillus acidoterrestris），是苹果浓缩汁生产中重要的目标控制微生物。耐热菌的耐热性强，能够经受酸性果汁加工中的巴氏杀菌过程而存活；存活下来的耐热菌即使在极低的浓度下，遇到合适的条件，也可在果汁中迅速生长繁殖而导致果汁感官品质的劣变。因此，国际贸易中一般要求每10克苹果浓缩汁中耐热菌的含量小于1。然而，对耐热菌的控制，直至目前仍无成熟可靠的方法。耐热菌超标是苹果浓缩汁生产中最为严重的质量问题之一，也是当前我国苹果浓缩汁行业产品出口中所遭遇的主要的技术壁垒之一，成为果汁生产的“瓶颈”。 耐热菌的有效控制，首先要求其得到快速而准确的检测。目前沿用的耐热菌检测方法为常规的培养检测法，耗时很长，一般需要4～5日才能出检测报告。检测结果的滞后性使之无法及时指导生产，无法及时向生产线反馈信息以采取相应的防范、控制与清洗措施。所以，苹果浓缩汁生产中迫切需要一种快速、准确的耐热菌检测方法。 PCR（聚合酶链反应）是体外快速扩增核酸的方法，它能使极微量的核酸在数小时之内扩增至原来的数百万倍以上。只要选择合适的引物，PCR就可特异性地大量扩增某一DNA片断至易检测水平。利用此原理，可以通过特异性地扩增某种微生物的基因片断而实现快速检测目的。 本实验选择了耐热菌的16S rDNA的高变区V2区和V4区某一段作为特异性引物的接合区域，以实现对此种微生物的特异的快速检测。通过实验研究，确定了苹果浓缩汁中耐热菌的PCR检测方法体系，并对此检测体系作了初步的应用及信度评价；还以此为基础，自制了耐热菌PCR快速诊断试剂盒，并对该试剂盒作了初步研究与应用评价。通过研究得到以下结论： 1．所选用的引物具有良好的种特异性。该引物可使耐热菌（酸土环状脂肪酸芽孢杆菌）特异性地扩增出294bp长度的DNA片断，而对其它近缘或远缘的各菌株都不产生任何扩增片断。 2．通过优化试验，得到本实验的PCR扩增最优化条件：50μL扩增体系中，Taq酶2U、Mg²⁺浓度2.0mmol／L；最佳退火温度58℃；循环程序为：94℃变性4min后进入PCR循环，94℃30S、58℃30S、72℃30S，扩增35个循环后72℃延伸补足5min。 3．最优化扩增程序下，PCR扩增呈阳性的耐热菌的基因组DNA最低量为 1刀pg，PCR扩增呈阳性的最低菌液浓度为 5刀x 103CFU／mL(水浴法破壁人 刀．为检出苹果汁中低浓度的耐热菌，在PCR反应之前必须进行预培养增殖。 最佳预培养条件为：402培养液，pH4刀，45’C通气振荡培养15h。 5．耐热菌DNA最佳提取方法：比较研究了CTAB法、SDS法、水浴法对 耐热菌DNA提取效率的影响，三种提取方法耐热菌DNA的得率依次略有降低， 差别不大；但前两种方法操作过程繁琐，费时较多，而水浴法单管操作，简便快 捷。综合考虑，以水浴法提取DNA最佳。 6．成功建立了苹果浓缩汁中耐热菌的PCR快速检测方法。该检测方法整体 体系为：10g苹果浓缩汁一无菌水适度稀释一小滤膜（直径中 25mm，膜孔 径 0．22 u m）针筒式滤器过滤一 小滤膜转至 402培养液中 一 70 C热处理 10min 一45℃气浴振荡培养15h一培养液12000rpm离心10min一沉淀转入小离心 管中 一水浴法裂解提取 DN A一 提取的 DNA进行 PCR扩增 一扩增产物凝 胶电泳检测一 结果判断。整个检测过程约需 18～20h。PCR法检测结果与 KFL 常规培养检测法结果完全相符。 7．自制了耐热菌的PCR诊断试剂盒。该试剂盒的信度评价、重复性评价及 有效期评价结果表明：其检测结果与常规检测方法结果一致，重复性好，有效期 至少为60d（更长的有效期待进一步考察），可考虑实践中的应用。更多还原

【Abstract】 Alicyclobacillus acidoterrestris, also called as thermophic acidophlic bacteria(TAB), is an important target micro-organism in quality control of apple juice concentrate(AJC). The spores of A. acidoterrestris are relatively heat resistant and can surive conventional pasteurisation treatment applied to AJC, and they can germinate and outgrow under suitable conditions causing spoilage. In the international trade of AJC, there are always demands for A. acidoterrestris being less than ICFU/10g AJC. A, acidoterrestris is one of the most serious quality problems in China AJC industry, for no feasible ways can control it at present.Therefore, rapid detection of such bacteria is essential for the quality control of AJC product. At present A. acidoterrestris is detected by plate count method, but it takes too much time, generally four to five days being required. Rapid and accurate detection methods are expected to develop.Polymerase chain reaction (PCR) is an elegent technique of creating copies of specific fragments of DNA, which rapidly amplifies a single DNA fragment into millions. The technology of PCR can be used to detect microorganisms quickly as long as the primers used are suitable.In this paper, two oligonucleotide primers were designed from the V2 and V4 regions of A. acidoterrestris’ 16S rDNA sequence. A PCR method for rapid and specific detecting of A. acidoterrestris is investigated, and its reliability is judged as well. Diagnostic kit for detecting of TAB is developed; its performance is also assessed by comparison with routine detecting method.The results of the studies are listed as follows:1. The primers used are strictly specific. Only A. acidoterrestris produces an intense band of 294bp after amplification; to the contrast, all the other bacteria strains can’t produce any bands during the same amplification.2. The optimum parameter for PCR reactionary system: Taq DNA polymerase 2U, Mg2+ 2.0mmol/L in 50L volume. And the annealing temperature is selected at 58 C. The PCR amplifying is done according to the following program: pre-denaturation at94 ℃ for 4min;35cyclesof94 ℃ for30S,58 ℃ for 30S,72℃ for 30S, final extension at72℃ for 5mhio3. The detection limit of the PCR method is Ipg total genomic DNA. And it can detect A, addoterrestris as low to 5.0+103CFU/mL.4. To detect A. addoterrestris in low concentrate, the enrichment is necessary. The optimum enrichment culture condition is : 402 medium, pH 4.0 at 45℃ for 15h.5. Three means for DNA extraction (CTAB SDS, water- boiling) are investigated. The boiling-water bath means is simple and effective, so it is choosed as the optimum DNA extracting approach.6.The complete PCR detecting system is: AJC 10g-diluted by sterilized water -filtered through membrane( b 25mm, pore size 0.22 m) - membrane translated to 402 medium-70℃ heat shock for 10 min-cultured at 45℃for 15h- centrifuge at 12000rpm for 10min-sendiment moved to microfuge-tube-DNA extracted-PCR amplifying-results analysis.The PCR detection can be done in 18-20 hours. The detecting results obtained by PCR method coincide with those of KFL’s.7. The self-designed diagnose kit performs well. The kit’s detecting results are reliable and repeatable. Its period of validity is at least 60 days (the maximum period of validity needs further research).更多还原