苹果浓缩汁中耐热菌的PCR方法快速检测研究
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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、Mg~(2+)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。
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 at
94 ℃ for 4min;35cyclesof94 ℃ for30S,58 ℃ for 30S,72℃ for 30S, final extension at72℃ for 5mhio
3. 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).
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、Mg~(2+)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。
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 at
94 ℃ for 4min;35cyclesof94 ℃ for30S,58 ℃ for 30S,72℃ for 30S, final extension at72℃ for 5mhio
3. 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).
引文
[1] 胡小松、李积宏等，现代果蔬汁加工工艺学，中国轻工业出版社，1995．
[2] 仇农学、肖旭霖、邓红，陕西省浓缩苹果汁行业可持续发展的战略思考，农业工程学报，2000，16(1)，122-124．
[3] 曾祥奎，黄斌，于娟，浓缩苹果汁主要技术指标控制的研究，饮料工业，2002，5 (1)，19-30．
[4] 中国食品土畜进出口商会苹果汁分会统计资料，2003年3月．
[5] 陕西省果业管理局，陕西果业，2001，16．
[6] 宁正祥，赵谋明，食品生物化学，华南理工大学出版社，1995．
[7] 易建华，仇农学等，树脂法生产无色浓缩苹果汁的初步研究，饮料工业，2001，4(4)，6-7．
[8] 王思新、焦中高等，树脂吸附法提高苹果浓缩清汁色值和透光率研究，果树学报，2001，1，35-38．
[9] 张欣、蔡同一等，苹果浓缩汁的后混浊，食品科学，1998，19(1)：22-24．
[10] 张建军、蔡同一等，减轻超滤苹果汁后浑浊的途径，食品工业科技，1992，3，8．
[11] 蔡静，浓缩苹果汁中甲胺磷农药残留规律及去除方法研究，西北农业大学1999届硕士学位毕业论文．
[12] 丁辰，苹果浓缩汁加工季节中棒曲霉素含量变化，中国果菜，2000，4，22．
[13] Jensen, N., Alicyclobacillus-a new challenge for the food industry, Food Australia, 1999, 51(1,2), 33-36.
[14] Borlinghaus, A.&Engle, R.,Alicyclobacillus incidene in commercial apple jiuce concentrate(AJC) supplies—Method development and validation, Fruit Processing, 1997, 7(7), 262-266.
[15] Baumgart, J., Menje, S., The impact of Alicyclobacillus acidoterrstris on the quality of juices and soft drinks, Fruit Processing, 2000, 10(7), 251-254.
[16] 姚培鑫，马小魁，浓缩果汁生产厂中嗜酸耐热菌的跟踪检测，饮料工业，2001，3，42-44．
[17] KFL 网页：http://www.kfl.com/atb.html
[18] 和致中等，高温菌生物学，科学出版社，2000
[19] McIntyre, S.,et al, Characteristics of an acidophilic Bacillus strain isolated from shelf-stable juices, J. Food Prot.,1995,58(3), 319-321.
[20] Previdi, M.P.,et al, Characterization of Alicyclobacillus,a spore-forming thermophilic acidophilic bacterium, Industria Conserve,1995,70(2),128-132.
[21] Walls, I., Chuyate, R., Alicyclobacillus--historical perspective and preliminary characterization study, Dairy, food and enviro. sanitation, 1998, 18(8), 499-503.
[22] 天津轻工业学院，无锡轻工业学院合编，食品工艺学(上册)，中国轻工业出版社，1995．
[23] Cerny, G. et al,Spoilage of fruit juice by Bacillus: isolation and characterisation of the spoiling microorganism, Z. Lebensm. Unters. Forch. 1984, 179: 224-227.
[24] Jensen, N., Alicyclobacillus in Australia, Food Australia, 2000, 52 (7), 33-36.
[25] Yamazaki,K.,Teduka, H.,Shinano, H., Isolation and Identification of Alicyclobacillus acidoterrestris from Acidic Beverages, Biosci. Biotech. Biochem., 1996, 60(3), 543-545.
[26] Walls, I. and Chuyate R., Spoilage of fruit juice by Alicyclobacillus acidoterrstris, Food Australia, 2000, 52(7), 286-291.
[27] Uchino, F. &Doi,s., Acido-thermophilic bacteria from thermal waters, Agric. Biol. Chem., 1967, 31, 817-822.
[28] Darland,G. &Brock, TD., Bacillus acidocaldarius sp. nov., an acidophilic thermophlic spore-forming bacterium, J. Gen.Microbiol., 1971, 67, 9-15.
[29] Deinhard, G.,Blanz, P.,Poralla, K.,Altan, E., Bacillus acidoterrestris sp. nov., a new thermotolerant acidophile isolated from different soils, System. Appl. Microbiolo., 1987,10 (1),47-53.
[30] Deinhard, O.,Saar, J.,Krischke, W.,Poralla, K., Bacillus cycloheptanicus sp. nov., a new thermoacidophile containing ω-cycloheptane fatty acids, System. Appl. Microbiol.,1987,10(1): 68-73.
[31] de Rosa, M.,et al, Cyclohexane fatty acids from a thermophilic bacterium, Chem. Comm.,1971,1334.
[32] Poralla,K.,Koenig, WA., The occurrence of ω-cycloheptane fatty acids in a thermo-acidophilic Bacillus, FEMS Microbiol. Lett.,1983,
16 (2-3), 303-306.
[33] Wisotzkey, J.D. et al., Comparative sequence analyses on the 16S rRNA (rDNA) of Bacillus acidocaldarius, Bacillus acidoterrestris, and Bacillus cycloheptanicus and proposal for creation of a new genus, Alicyclobacillus gen. nov., Int. J. Syst. Bacteriol.,1992,42,263-269.
[34] Abuquerque, L., Alicyclobacillus hesperidum sp. nov. and a related genomic species from solfataric soils of S(?)o Miguel in the Azores, Int. J. Syst. Evol. Microbiol.,2000,50,451-457.
[35] Goto, K., et al, Alicyclobacillus herbarius sp. nov., a novel bacterium containing omega cycloheptane fatty acids, isolated from herbal tea, Int. J. Syst. Evol. Mierobiol.,2002,52, 109-113
[36] Cerny, G., Duong, H-A., Hennlich, W., Miller, S., Alicyclobacillus acidoterrestris:influence of oxygen content on growth in fruit juices, Food Australia, 2000, 52(7), 289-291.
[37] Pontius, A.J., et al, Heat resistance of Alicyclobacillus acidoterrstris spores as affected by various pH values and organic acids, J. Food Prot., 1998, 61, 41-46.
[38] Nicolaus, B.,Improta, R.,et al., Alicyclobacilli from an unexplored geothermal soil in Antarctica: Mount Rittmann, Polar Biol.,1998, 19:133-141
[39] Eguchi, S.Y.,et al, Acidothermophilic sporeforming bacteria (ATSB) in orange juice: Delection methods,ecology, and involvement in the deterioration of fruit juices-Report of the research project, Fruit Processing, 2001,11, 12-18.
[40] Splittstoesser, D.F., Churey, J.J., Lee, C.Y., Growth characteristics of aciduric sporeforming bacilli isolated from fruit juices, J. Food Prot., 1994,57(12),1080-1083
[41] Eiroa, M.N.U.,Junqueira, V.C.A.,Schmidt, F.L.,Alicyclobacillus in orange juice:occurrence and heat resistance of spores, J. Food Frot.,1999,62(8), 883-886.
[42] Murakami, M., Tedzuka, H., Yamazaki, K., Thermal resistance of Alicyclobacillus acidoterrestris spores in difference buffers and pH, Food microbiology, 1998, 15(6), 577-582.
[43] F. Silva F.M. et al.,Thermal inactivation of Mlicyclobacillus acidoterrestris Spores under different temprature, soluble solids and pH conditions for the design of fruit processes, Int. J. Food Microbiol.,1999,51:95-103.
[44] Silva, F. V. M. and Gibbs, P., Alicyclobacillus acidoterrestris spores in fruit products and design of pasteurization processes, Trends in Food Science &Technology, 2001,12:68-74
[45] Splittstoesser, D.F.,Lee, C.Y.,churey, J.J., Control of Alicyclobacillus in the juice industry, Dairy, Food and Environmental Sanitation, 1998,18(9), 585-587.
[46] Yamazaki, K.,Isoda, C.,Teduka, H.,Kawai,Y.&Shinano, H.,Thermal resistance and prevention of spoilage bacterium, Alicyclobacillus acidoterrestris, in acidic beverages, Nippon Shokuhin Kagaku Kogaku Kaishi, 1997(44),905-911
[47] Kannenberg, E.,Blume,A.&Poralla, K.,Properties of ω-cycloheptane fatty acids in membranes, FEBS Lest., 1984(172),331-334.
[48] Whitfirld, F.B.,Last, J.H., et al, 2,6-dibromophenol: the cause of an iodoform-like off-flavour in some Australian crustacea, Journal of the science of food and agriculture, 1988,46(1),29-42.
[49] Orr, R.V., et al, Detection of guaiacol produced by Alicyclobacillus acidoterrestris in apple juice by sensory and chromatographic analyses, and comparison with spore and vegetative cell populations, J. Food Prot., 2000, 63 (11),1517-1522.
[50] Luscardi, C. et al, Ability of Alicyclobacillus strains to spoil fruit juice and nectars, Indudtria Conserve, 2000,75(2),151-161.
[51] Pettipher,G. L., Osmundson, M.E., Murphy, J.M., Methods for the detection and enumeration of Alicyclobaeillus acidoterrestris and investigation of growth and production of taint in fruit juice and fruit juice containing drinks, Letters in Applied Microbiology, 1997, 24(3), 185-189
[52] Jensen, N., Thermotolerant aerobic spore formers--Alicyclobacillus, 2001(unpublished).
[53] Pettipher,G.L.,Osmundson, M. E.,Methods for the detection, enumeration
and identification of Alicyclobacillus acidoterrstris, Food Australia, 2000, 52(7), 293-295.
[54] Komitopoulou, E., et al, Alicyclobacillus acidoterrestris in fruit juices and its control by nisin, International journal of food science & technology, 1999, 34 (1), 81-85.
[55] Yamazaki, K., et al, Use of nisin for inhibition of Alicyclobacillus acidoterrestris in acidic drinks, Food Microbiology, 2000 17(3):315-320.
[56] Yamazaki,K., et al, Thermal resistence and prevention of spoilage bacterium, Alicyclobacillus acidoterrestris, in acidic beverages, Journal of the Japanese Society of Food Science and Technology, 1997,44(12):905-911.
[57] Lee, S-Y.,Dougherty, RH.,Kang, D-H.,Inhibitory Effects of High Pressure and Heat on Alicyclobacillus acidoterrestris Spores in Apple Juice, Appl. Environ. Microbiol., 2002,68(8),4158-4161.
[58] Orr, R.V., Beuchat, L.R.,Efficacy of disinfectants in killing spores of Alicyclobacillus acidoterrestris and performance of media for supporting colony development by survivors, Journal of food protection, 2000, 63(8), 1117-1122.
[59] 王思新等，浓缩苹果汁加工中耐热菌的分析与控制，食品科学，2000，21(9)，54-56．
[60] 常玉华，仇农学，苹果浓缩汁中的耐热菌—特性、检测与控制，陕西食品工业，2002，2，7-10．
[61] 杨苏声，细菌分类学，中国农业大学出版社，1997．
[62] Yamazaki, K.,Teduka, H.,Inoue, N.,Shinano, H.,Specific primers for detection of Alicyclobacillus acidoterrestris by RT-PCR,Lett. Appl. Microbiol.,1996,23, (5),350-354.
[63] 迪芬巴赫C．W．，PCR技术实验指南，黄培堂译，科学出版社，2000．
[64] 陈启民，王金忠，耿运琪，分子生物学，南开大学出版社，2001．
[65] 奥斯伯F．等，精编分子生物学实验指南，颜子颖等译，科学出版社，1998．
[66] 萨姆布鲁克J．，弗里厅E．F．，曼尼阿蒂斯，分子克隆实验指南(第二版)，金冬雁，黎孟枫等译，科学出版社，1992．
[67] 潘耀谦，金春彬，聚合酶链反应(PCR)技术体系研究进展，动物医学进展，
1999, 20(4), 11-17.
[68] Sano, T., et al, Immuno-PCR:Very sensitive antigen detection by means of specific antibody-DNA conjugates, Science, 1992, 258: 120～122.
[69] 曾常茜，晏舒，胡景新，免疫PCR的方法学研究进展及临床应用，北华大学学报(自然科学版)，2002，1，30-33．
[70] 张莉萍，郑佐华，毛裕民，耐热逆转录套式PCR法检测丙型肝炎病毒，中华预防医学杂志 1998，3，164．
[71] 张放等，应用套式PCR检测嗜肺军团杆菌的实验研究，中国医科大学学报，1995，6，575-577．
[72] 李君文等，套式反转录PCR检测水中肠道病毒，中国公共卫生，1996，9，406-407．
[73] 袁长青，李君文，一步单管反转录PCR快速检测水中甲型肝炎病毒，中国卫生检验杂志，1999，4，243-345．
[74] 任少堂等，多重PCR技术在医学诊断中的应用与发展，临床检验杂志，1995，13(1)：45．
[75] Ombrouck C., Ciceron L., Biligui S., et al., Specif-ic PCR assay for direct detection of intestinal microsporidia Enterocytozoon bieneusi and Encephalitozoonintestinalis infecal specimens from human immunodeficiency virus-infected patients, J. Clin. Microbiol.,1997, 35(3):652～655.
[76] Asimk, B., et al, Detection of coliform bacteria in water by polymerase chain reaction and gene probes, Appl. Environ. Mcrobiol. 1990, 56(2), 307.
[77] 肖文华，热启动PCR，国外医学生物化学与检验学分册，1996，17(2)，68．
[78] Hayashi, K., Orita, M., Suzuki, Y., Sekiya, T., Use of labeled primers in polymerase chain reaction (LP-PCR) for a rapid detection of the product, Nucleic Acids Research, 1989, 17(9),3605.
[79] 林玲，定量PCR技术的研究进展，国外医学遗传学分册，1999，22(3)，116-120．
[80] 张学敏，王宜强，靶向新基因的分子科隆策略—理论与方法，军事医学科学院出版社，1999．
[81] 李君文，晁福寰，致病微生物PCR检测方法研究进展，中国卫生检验杂志，1997，7(5)，313-318．
[82] 叶怀庄，吴丽丽，PCR技术在卫生微生物检测中的应用，中国卫生检验杂志，
1996，4，220-223．
[83] 胡稳奇，张志光，PCR技术在环境微生物检测中的应用，环境科学，1994，4，80-83．
[84] 李平兰，PCR技术及其在食品微生物检测中的应用，食品科学，1998，19(7)，3-5．
[85] 李凤义，PCR检测水中致病微生物的研究进展，工业卫生与职业病，1996，(6)，379-382．
[86] Asmk B.,et al, Polymerase chain reaction-Gene probe setection of microorganisms by using filter—concentrated samples, Appl. Environ. Mcrobiol. 1991, 57(12),3529.
[87] 李谦，李雅青，提高PCR反应特异性的几点策略，临沂师范学院学报，2001，4，56-57．
[88] 袁长青，李平，PCR反应条件的优化，中国公共卫生，1999，15(3)，255-256．
[89] 张龙翔，张庭芳，李令媛，生化实验方法和技术(第二版)，高等教育出版社，1997．
[90] 曹泽虹，李勇，用PCR法快速测定食物中毒病原菌，微生物学通报，2001，4：72-76．
[91] Abravaya K., et al, Performance of a Multiplex Qualitative PCR LCx Assay for Detection of Human Immunodeficiency Virus Type 1 (HIV-1) Group M Subtypes, Group O, and HIV-2, J. Clin. Microbiol., 2000, 38(2): 716-723.
[92] 王静等，多重PCR快速检测食品中肠出血性大肠杆菌，卫生研究，2001，30(5)，310-312．
[93] Kong, RYC., et al, A Sensitive and Versatile Multiplex PCR System for the Rapid Detection of Enterotoxigenic (ETEC), Enterohaemorrhagic (EHEC) and Enteropathogenic (EPEC) Strains of Escherichia coli, Marine Pollution Bulletin, 1999,38(12), 1207-1215.
[94] 李景鹏等，应用聚合酶链反应快速检测沙门氏菌，东北农业大学学报，1997，28(1)，79-83．
[95] 杨百亮等，快速检测牛奶中单核细胞增多症李氏菌PCR试剂盒的研制，中国公共卫生，1994，10(7)，304-305．
[96] 王月玲等，增菌PCR法检测小鼠沙门氏菌，上海实验动物科学，2000，20(3)：144-147．
[97] Karunasagar, I.,et al, Rapid polymerase chain reaction method for detection of Kanagawa positive Vibrio parahaemolyticus in seafoods, Int. J. Food Microbiol.,1996,31,317-323.
[98] 周志江等，用PCR检测副溶血性弧菌耐热溶血素基因，中国兽医学报，1996，3，251-255．
[99] 封幼玲等，应用PCR技术快速检测食品中副溶血性弧菌，中国卫生检验杂志，1997，4：241-242．
[100] 云泓若，李月琴，周天鸿，PCR技术检测金黄色葡萄球菌肠毒素D基因，暨南大学学报(自然科学与医学版)，1999，5，84-87．
[101] Tsen, H.Y., Chen, T.R., Use of the polymerase chain reaction for specific detection of type A, D and E enterotoxigenic Staphylococcus aureus in foods, Applied microbiology and biotechnology, 1992, 37(5), 685-690.
[102] Khan, M.A., Detection of Staphylococcus aureus in milk by use of polymerase chain reaction analysis, American journal of veterinary research, 1998, 59(7), 807-813.
[103] 寇运同，马洪明，刘晨光，用PCR技术快速检测食品中的单核细胞增生性李斯特菌，食品科学，2001，(22)5，52-55．
[104] Bansal, N.S., Development of apolymerase chain reaction assay for the detection of Listeria monocytogenes in foods, Letters in applied microbiology, 1996, 22 (5), 353-356.
[105] Makino, S., Okado, Y.,Maruyama, T., A new method for direct detection of Listeria monocytogenes from foods by PCR, Appl. Environ. Microbiol., 1995, 61(10), 3745-3747.
[106] 王颖群，严共华，雷祚荣，PCR检测食品标本中A、B、E和F型肉毒梭菌，中国公共卫生，1997，9：560-561．
[107] Kaufmann, P., Pfefferkorn, A.,Teuber, M., Meile, L., Identification and quantification of Bifidobacterium species isolated from food with genus-specific 16S rRNA-targeted probes by colony hybridization and PCR, Appl. Environ. Microbiol., 1997, 63(4),1268-73.
[108] DSMZ: http://www.dsmz.de/media/med402.htm.
[109] 周德庆，微生物学教程，高等教育出版社，1993．
[110] 沈萍，范秀容，李广武，微生物学实验(第三版)，高等教育出版社，1999
[111] 焦振泉，刘秀梅，16s rRNA 序列同源性分析与细菌系统分类鉴定，国外医学卫生学分册，1998，25(1)，12-16．
[112] Meier, A., et al, Elimination of contaminating DNA within polymerase chain reaction reagents: Implications for a general approach to detection of uncultured pathogens, J. Clin. Microbiol., 1993, 31(3), 646-652.
[2] 仇农学、肖旭霖、邓红，陕西省浓缩苹果汁行业可持续发展的战略思考，农业工程学报，2000，16(1)，122-124．
[3] 曾祥奎，黄斌，于娟，浓缩苹果汁主要技术指标控制的研究，饮料工业，2002，5 (1)，19-30．
[4] 中国食品土畜进出口商会苹果汁分会统计资料，2003年3月．
[5] 陕西省果业管理局，陕西果业，2001，16．
[6] 宁正祥，赵谋明，食品生物化学，华南理工大学出版社，1995．
[7] 易建华，仇农学等，树脂法生产无色浓缩苹果汁的初步研究，饮料工业，2001，4(4)，6-7．
[8] 王思新、焦中高等，树脂吸附法提高苹果浓缩清汁色值和透光率研究，果树学报，2001，1，35-38．
[9] 张欣、蔡同一等，苹果浓缩汁的后混浊，食品科学，1998，19(1)：22-24．
[10] 张建军、蔡同一等，减轻超滤苹果汁后浑浊的途径，食品工业科技，1992，3，8．
[11] 蔡静，浓缩苹果汁中甲胺磷农药残留规律及去除方法研究，西北农业大学1999届硕士学位毕业论文．
[12] 丁辰，苹果浓缩汁加工季节中棒曲霉素含量变化，中国果菜，2000，4，22．
[13] Jensen, N., Alicyclobacillus-a new challenge for the food industry, Food Australia, 1999, 51(1,2), 33-36.
[14] Borlinghaus, A.&Engle, R.,Alicyclobacillus incidene in commercial apple jiuce concentrate(AJC) supplies—Method development and validation, Fruit Processing, 1997, 7(7), 262-266.
[15] Baumgart, J., Menje, S., The impact of Alicyclobacillus acidoterrstris on the quality of juices and soft drinks, Fruit Processing, 2000, 10(7), 251-254.
[16] 姚培鑫，马小魁，浓缩果汁生产厂中嗜酸耐热菌的跟踪检测，饮料工业，2001，3，42-44．
[17] KFL 网页：http://www.kfl.com/atb.html
[18] 和致中等，高温菌生物学，科学出版社，2000
[19] McIntyre, S.,et al, Characteristics of an acidophilic Bacillus strain isolated from shelf-stable juices, J. Food Prot.,1995,58(3), 319-321.
[20] Previdi, M.P.,et al, Characterization of Alicyclobacillus,a spore-forming thermophilic acidophilic bacterium, Industria Conserve,1995,70(2),128-132.
[21] Walls, I., Chuyate, R., Alicyclobacillus--historical perspective and preliminary characterization study, Dairy, food and enviro. sanitation, 1998, 18(8), 499-503.
[22] 天津轻工业学院，无锡轻工业学院合编，食品工艺学(上册)，中国轻工业出版社，1995．
[23] Cerny, G. et al,Spoilage of fruit juice by Bacillus: isolation and characterisation of the spoiling microorganism, Z. Lebensm. Unters. Forch. 1984, 179: 224-227.
[24] Jensen, N., Alicyclobacillus in Australia, Food Australia, 2000, 52 (7), 33-36.
[25] Yamazaki,K.,Teduka, H.,Shinano, H., Isolation and Identification of Alicyclobacillus acidoterrestris from Acidic Beverages, Biosci. Biotech. Biochem., 1996, 60(3), 543-545.
[26] Walls, I. and Chuyate R., Spoilage of fruit juice by Alicyclobacillus acidoterrstris, Food Australia, 2000, 52(7), 286-291.
[27] Uchino, F. &Doi,s., Acido-thermophilic bacteria from thermal waters, Agric. Biol. Chem., 1967, 31, 817-822.
[28] Darland,G. &Brock, TD., Bacillus acidocaldarius sp. nov., an acidophilic thermophlic spore-forming bacterium, J. Gen.Microbiol., 1971, 67, 9-15.
[29] Deinhard, G.,Blanz, P.,Poralla, K.,Altan, E., Bacillus acidoterrestris sp. nov., a new thermotolerant acidophile isolated from different soils, System. Appl. Microbiolo., 1987,10 (1),47-53.
[30] Deinhard, O.,Saar, J.,Krischke, W.,Poralla, K., Bacillus cycloheptanicus sp. nov., a new thermoacidophile containing ω-cycloheptane fatty acids, System. Appl. Microbiol.,1987,10(1): 68-73.
[31] de Rosa, M.,et al, Cyclohexane fatty acids from a thermophilic bacterium, Chem. Comm.,1971,1334.
[32] Poralla,K.,Koenig, WA., The occurrence of ω-cycloheptane fatty acids in a thermo-acidophilic Bacillus, FEMS Microbiol. Lett.,1983,
16 (2-3), 303-306.
[33] Wisotzkey, J.D. et al., Comparative sequence analyses on the 16S rRNA (rDNA) of Bacillus acidocaldarius, Bacillus acidoterrestris, and Bacillus cycloheptanicus and proposal for creation of a new genus, Alicyclobacillus gen. nov., Int. J. Syst. Bacteriol.,1992,42,263-269.
[34] Abuquerque, L., Alicyclobacillus hesperidum sp. nov. and a related genomic species from solfataric soils of S(?)o Miguel in the Azores, Int. J. Syst. Evol. Microbiol.,2000,50,451-457.
[35] Goto, K., et al, Alicyclobacillus herbarius sp. nov., a novel bacterium containing omega cycloheptane fatty acids, isolated from herbal tea, Int. J. Syst. Evol. Mierobiol.,2002,52, 109-113
[36] Cerny, G., Duong, H-A., Hennlich, W., Miller, S., Alicyclobacillus acidoterrestris:influence of oxygen content on growth in fruit juices, Food Australia, 2000, 52(7), 289-291.
[37] Pontius, A.J., et al, Heat resistance of Alicyclobacillus acidoterrstris spores as affected by various pH values and organic acids, J. Food Prot., 1998, 61, 41-46.
[38] Nicolaus, B.,Improta, R.,et al., Alicyclobacilli from an unexplored geothermal soil in Antarctica: Mount Rittmann, Polar Biol.,1998, 19:133-141
[39] Eguchi, S.Y.,et al, Acidothermophilic sporeforming bacteria (ATSB) in orange juice: Delection methods,ecology, and involvement in the deterioration of fruit juices-Report of the research project, Fruit Processing, 2001,11, 12-18.
[40] Splittstoesser, D.F., Churey, J.J., Lee, C.Y., Growth characteristics of aciduric sporeforming bacilli isolated from fruit juices, J. Food Prot., 1994,57(12),1080-1083
[41] Eiroa, M.N.U.,Junqueira, V.C.A.,Schmidt, F.L.,Alicyclobacillus in orange juice:occurrence and heat resistance of spores, J. Food Frot.,1999,62(8), 883-886.
[42] Murakami, M., Tedzuka, H., Yamazaki, K., Thermal resistance of Alicyclobacillus acidoterrestris spores in difference buffers and pH, Food microbiology, 1998, 15(6), 577-582.
[43] F. Silva F.M. et al.,Thermal inactivation of Mlicyclobacillus acidoterrestris Spores under different temprature, soluble solids and pH conditions for the design of fruit processes, Int. J. Food Microbiol.,1999,51:95-103.
[44] Silva, F. V. M. and Gibbs, P., Alicyclobacillus acidoterrestris spores in fruit products and design of pasteurization processes, Trends in Food Science &Technology, 2001,12:68-74
[45] Splittstoesser, D.F.,Lee, C.Y.,churey, J.J., Control of Alicyclobacillus in the juice industry, Dairy, Food and Environmental Sanitation, 1998,18(9), 585-587.
[46] Yamazaki, K.,Isoda, C.,Teduka, H.,Kawai,Y.&Shinano, H.,Thermal resistance and prevention of spoilage bacterium, Alicyclobacillus acidoterrestris, in acidic beverages, Nippon Shokuhin Kagaku Kogaku Kaishi, 1997(44),905-911
[47] Kannenberg, E.,Blume,A.&Poralla, K.,Properties of ω-cycloheptane fatty acids in membranes, FEBS Lest., 1984(172),331-334.
[48] Whitfirld, F.B.,Last, J.H., et al, 2,6-dibromophenol: the cause of an iodoform-like off-flavour in some Australian crustacea, Journal of the science of food and agriculture, 1988,46(1),29-42.
[49] Orr, R.V., et al, Detection of guaiacol produced by Alicyclobacillus acidoterrestris in apple juice by sensory and chromatographic analyses, and comparison with spore and vegetative cell populations, J. Food Prot., 2000, 63 (11),1517-1522.
[50] Luscardi, C. et al, Ability of Alicyclobacillus strains to spoil fruit juice and nectars, Indudtria Conserve, 2000,75(2),151-161.
[51] Pettipher,G. L., Osmundson, M.E., Murphy, J.M., Methods for the detection and enumeration of Alicyclobaeillus acidoterrestris and investigation of growth and production of taint in fruit juice and fruit juice containing drinks, Letters in Applied Microbiology, 1997, 24(3), 185-189
[52] Jensen, N., Thermotolerant aerobic spore formers--Alicyclobacillus, 2001(unpublished).
[53] Pettipher,G.L.,Osmundson, M. E.,Methods for the detection, enumeration
and identification of Alicyclobacillus acidoterrstris, Food Australia, 2000, 52(7), 293-295.
[54] Komitopoulou, E., et al, Alicyclobacillus acidoterrestris in fruit juices and its control by nisin, International journal of food science & technology, 1999, 34 (1), 81-85.
[55] Yamazaki, K., et al, Use of nisin for inhibition of Alicyclobacillus acidoterrestris in acidic drinks, Food Microbiology, 2000 17(3):315-320.
[56] Yamazaki,K., et al, Thermal resistence and prevention of spoilage bacterium, Alicyclobacillus acidoterrestris, in acidic beverages, Journal of the Japanese Society of Food Science and Technology, 1997,44(12):905-911.
[57] Lee, S-Y.,Dougherty, RH.,Kang, D-H.,Inhibitory Effects of High Pressure and Heat on Alicyclobacillus acidoterrestris Spores in Apple Juice, Appl. Environ. Microbiol., 2002,68(8),4158-4161.
[58] Orr, R.V., Beuchat, L.R.,Efficacy of disinfectants in killing spores of Alicyclobacillus acidoterrestris and performance of media for supporting colony development by survivors, Journal of food protection, 2000, 63(8), 1117-1122.
[59] 王思新等，浓缩苹果汁加工中耐热菌的分析与控制，食品科学，2000，21(9)，54-56．
[60] 常玉华，仇农学，苹果浓缩汁中的耐热菌—特性、检测与控制，陕西食品工业，2002，2，7-10．
[61] 杨苏声，细菌分类学，中国农业大学出版社，1997．
[62] Yamazaki, K.,Teduka, H.,Inoue, N.,Shinano, H.,Specific primers for detection of Alicyclobacillus acidoterrestris by RT-PCR,Lett. Appl. Microbiol.,1996,23, (5),350-354.
[63] 迪芬巴赫C．W．，PCR技术实验指南，黄培堂译，科学出版社，2000．
[64] 陈启民，王金忠，耿运琪，分子生物学，南开大学出版社，2001．
[65] 奥斯伯F．等，精编分子生物学实验指南，颜子颖等译，科学出版社，1998．
[66] 萨姆布鲁克J．，弗里厅E．F．，曼尼阿蒂斯，分子克隆实验指南(第二版)，金冬雁，黎孟枫等译，科学出版社，1992．
[67] 潘耀谦，金春彬，聚合酶链反应(PCR)技术体系研究进展，动物医学进展，
1999, 20(4), 11-17.
[68] Sano, T., et al, Immuno-PCR:Very sensitive antigen detection by means of specific antibody-DNA conjugates, Science, 1992, 258: 120～122.
[69] 曾常茜，晏舒，胡景新，免疫PCR的方法学研究进展及临床应用，北华大学学报(自然科学版)，2002，1，30-33．
[70] 张莉萍，郑佐华，毛裕民，耐热逆转录套式PCR法检测丙型肝炎病毒，中华预防医学杂志 1998，3，164．
[71] 张放等，应用套式PCR检测嗜肺军团杆菌的实验研究，中国医科大学学报，1995，6，575-577．
[72] 李君文等，套式反转录PCR检测水中肠道病毒，中国公共卫生，1996，9，406-407．
[73] 袁长青，李君文，一步单管反转录PCR快速检测水中甲型肝炎病毒，中国卫生检验杂志，1999，4，243-345．
[74] 任少堂等，多重PCR技术在医学诊断中的应用与发展，临床检验杂志，1995，13(1)：45．
[75] Ombrouck C., Ciceron L., Biligui S., et al., Specif-ic PCR assay for direct detection of intestinal microsporidia Enterocytozoon bieneusi and Encephalitozoonintestinalis infecal specimens from human immunodeficiency virus-infected patients, J. Clin. Microbiol.,1997, 35(3):652～655.
[76] Asimk, B., et al, Detection of coliform bacteria in water by polymerase chain reaction and gene probes, Appl. Environ. Mcrobiol. 1990, 56(2), 307.
[77] 肖文华，热启动PCR，国外医学生物化学与检验学分册，1996，17(2)，68．
[78] Hayashi, K., Orita, M., Suzuki, Y., Sekiya, T., Use of labeled primers in polymerase chain reaction (LP-PCR) for a rapid detection of the product, Nucleic Acids Research, 1989, 17(9),3605.
[79] 林玲，定量PCR技术的研究进展，国外医学遗传学分册，1999，22(3)，116-120．
[80] 张学敏，王宜强，靶向新基因的分子科隆策略—理论与方法，军事医学科学院出版社，1999．
[81] 李君文，晁福寰，致病微生物PCR检测方法研究进展，中国卫生检验杂志，1997，7(5)，313-318．
[82] 叶怀庄，吴丽丽，PCR技术在卫生微生物检测中的应用，中国卫生检验杂志，
1996，4，220-223．
[83] 胡稳奇，张志光，PCR技术在环境微生物检测中的应用，环境科学，1994，4，80-83．
[84] 李平兰，PCR技术及其在食品微生物检测中的应用，食品科学，1998，19(7)，3-5．
[85] 李凤义，PCR检测水中致病微生物的研究进展，工业卫生与职业病，1996，(6)，379-382．
[86] Asmk B.,et al, Polymerase chain reaction-Gene probe setection of microorganisms by using filter—concentrated samples, Appl. Environ. Mcrobiol. 1991, 57(12),3529.
[87] 李谦，李雅青，提高PCR反应特异性的几点策略，临沂师范学院学报，2001，4，56-57．
[88] 袁长青，李平，PCR反应条件的优化，中国公共卫生，1999，15(3)，255-256．
[89] 张龙翔，张庭芳，李令媛，生化实验方法和技术(第二版)，高等教育出版社，1997．
[90] 曹泽虹，李勇，用PCR法快速测定食物中毒病原菌，微生物学通报，2001，4：72-76．
[91] Abravaya K., et al, Performance of a Multiplex Qualitative PCR LCx Assay for Detection of Human Immunodeficiency Virus Type 1 (HIV-1) Group M Subtypes, Group O, and HIV-2, J. Clin. Microbiol., 2000, 38(2): 716-723.
[92] 王静等，多重PCR快速检测食品中肠出血性大肠杆菌，卫生研究，2001，30(5)，310-312．
[93] Kong, RYC., et al, A Sensitive and Versatile Multiplex PCR System for the Rapid Detection of Enterotoxigenic (ETEC), Enterohaemorrhagic (EHEC) and Enteropathogenic (EPEC) Strains of Escherichia coli, Marine Pollution Bulletin, 1999,38(12), 1207-1215.
[94] 李景鹏等，应用聚合酶链反应快速检测沙门氏菌，东北农业大学学报，1997，28(1)，79-83．
[95] 杨百亮等，快速检测牛奶中单核细胞增多症李氏菌PCR试剂盒的研制，中国公共卫生，1994，10(7)，304-305．
[96] 王月玲等，增菌PCR法检测小鼠沙门氏菌，上海实验动物科学，2000，20(3)：144-147．
[97] Karunasagar, I.,et al, Rapid polymerase chain reaction method for detection of Kanagawa positive Vibrio parahaemolyticus in seafoods, Int. J. Food Microbiol.,1996,31,317-323.
[98] 周志江等，用PCR检测副溶血性弧菌耐热溶血素基因，中国兽医学报，1996，3，251-255．
[99] 封幼玲等，应用PCR技术快速检测食品中副溶血性弧菌，中国卫生检验杂志，1997，4：241-242．
[100] 云泓若，李月琴，周天鸿，PCR技术检测金黄色葡萄球菌肠毒素D基因，暨南大学学报(自然科学与医学版)，1999，5，84-87．
[101] Tsen, H.Y., Chen, T.R., Use of the polymerase chain reaction for specific detection of type A, D and E enterotoxigenic Staphylococcus aureus in foods, Applied microbiology and biotechnology, 1992, 37(5), 685-690.
[102] Khan, M.A., Detection of Staphylococcus aureus in milk by use of polymerase chain reaction analysis, American journal of veterinary research, 1998, 59(7), 807-813.
[103] 寇运同，马洪明，刘晨光，用PCR技术快速检测食品中的单核细胞增生性李斯特菌，食品科学，2001，(22)5，52-55．
[104] Bansal, N.S., Development of apolymerase chain reaction assay for the detection of Listeria monocytogenes in foods, Letters in applied microbiology, 1996, 22 (5), 353-356.
[105] Makino, S., Okado, Y.,Maruyama, T., A new method for direct detection of Listeria monocytogenes from foods by PCR, Appl. Environ. Microbiol., 1995, 61(10), 3745-3747.
[106] 王颖群，严共华，雷祚荣，PCR检测食品标本中A、B、E和F型肉毒梭菌，中国公共卫生，1997，9：560-561．
[107] Kaufmann, P., Pfefferkorn, A.,Teuber, M., Meile, L., Identification and quantification of Bifidobacterium species isolated from food with genus-specific 16S rRNA-targeted probes by colony hybridization and PCR, Appl. Environ. Microbiol., 1997, 63(4),1268-73.
[108] DSMZ: http://www.dsmz.de/media/med402.htm.
[109] 周德庆，微生物学教程，高等教育出版社，1993．
[110] 沈萍，范秀容，李广武，微生物学实验(第三版)，高等教育出版社，1999
[111] 焦振泉，刘秀梅，16s rRNA 序列同源性分析与细菌系统分类鉴定，国外医学卫生学分册，1998，25(1)，12-16．
[112] Meier, A., et al, Elimination of contaminating DNA within polymerase chain reaction reagents: Implications for a general approach to detection of uncultured pathogens, J. Clin. Microbiol., 1993, 31(3), 646-652.