6 2 Vol. 6 No. 2 2015 2 Journal of Food Safety and Quality Feb., 2015 肖 剑 1, 陈秀云 2, 梁美丹 1, 陈楷 1, 洪红 1, 焦红 3* (1., 510410; 2., 510663; 3., 510623) 摘要 : 目的 (simultaneous amplification and testing, SAT) 方法, SAT, 结果 4 SAT 100%, ; (94.2%)(99.5%) (100%)(100%), 9.6%, 94% 结论 SAT, 4 关键词 : SAT; ; ; Simultaneous amplification and testing application in detection of foodborne pathogenic microorganisms XIAO Jian 1, CHEN Xiu-Yun 2, LIANG Mei-Dan 1, CHEN Kai 1, HONG Hong 1, JIAO Hong 3* (1.Guangzhou Institute of Food Inspection, Guangzhou 510410, China; 2. Guangzhou Yi-Jian Biomedical Technology Development Co. Ltd, Guangzhou 510663, China 3. Guangdong Entry-Exit Inspection and Quarantine Bureau, Guangzhou 510623, China) ABSTRACT: Objective To evaluate the application value of simultaneous amplification and testing (SAT) in detection of foodborne pathogenic microorganisms. Methods The sensitivity, specificity, false positive rate, false negative rate and accuracy of Salmonella spp., Staphylococcus aureus, Vibrio parahaemolyticus and Listeria monocytogenes were studied by using the methods of national standards and molecular sequencing. Meanwhile, the significant difference of positive rate was analyzed by the chi-square. Results The sensitivity of four pathogens were all 100%, and there were no false negative and misdetection rate. The specificity of Salmonella spp., Staphylococcus aureus, Vibrio parahaemolyticus and Listeria monocytogenes were 94.2%, 99.5%, 100%, and 100% respectively, and they all conformed to the requirements of the test. The false positive rates were all below 9.6% and the accuracy rates were superior to the standard (94%). Conclusion It is feasible to detect the four pathogens above with SAT for its high sensitivity, specificity and accuracy. KEY WORDS: simultaneous amplification and testing; food detection; sensitivity; specificity * 通讯作者 :,, E-mail: jhciq1228@163.com *Corresponding author: JIAO Hong, Professor, Guangdong Entry-Exit Inspection and Quarantine Bureau, No.66, Zhujiang New City Huacheng Avenue, Guangzhou 510623, China. E-mail: jhciq1228@163.com
2, : 683 1 引言,, 30% [1],,, [2] 2013 ( 2014 15 ), 32.2%,, 60.4%,, [3],, ELISA [4] [5] [6] [7], DNA PCR [8,9], [10],,, (simultaneous amplification and testing, SAT),,, [11] [12] [13] [14] SAT (2~12 h),,,, SAT (Salmonella spp.) (Staphylococcus aureus) (Vibrio parahaemolyticus)(listeria monocytogenes), 4 SAT, SAT 2 材料与方法 2.1 样品, 1 表 1 试验用样品种类 批次及检验项目 Table 1 The sample types, numbers and inspection items () 1 ( ) 30 2 () 30 3 (() ( ) ) 30 4 () 30 5 30 6 ( ) 30 7 () 30 8 ( ( ) ) 30 9 (() 30 10 ( ( )) 30 11 30 () 330 330 240 60 30
684 6 2.2 主要培养基及试剂 BPW (, ); 7.5% (); 3(); (); Baird-Parker ; LB (); (, ); ( ); SAT ( ); (()) 2.3 主要试验仪器 ( SP250); ( GR85DR); (ThermoFisher1300); VITEK2 ( ); (AB MagMAXTM Express 96); PCR (ABI 7500FAST) 2.4 试验方法及原理 2.4.1 国标法 GB4789.4-2010 [15] GB4789.10-2010 [16] GB4789.7-2013 [17] GB4789.30-2010 [18] 2.4.2 SAT 法 2.4.2.1 SAT 2.4.1, 200 μl, 10 μl, 200 μl 100 μl (), 60 5 min, 10 min, ( ), RNA, ;, 30 μl PCR, 10 μl SAT, PCR,, 2.4.2.2 SAT, ( ), NCBI(http://www.ncbi.nlm.nih.gov/Genbank), Blast 2.4.3 统计分析 2.4.3.1, SAT, [19] : 98%, 90.4%, <2%, <9.6%, 94%, <3.84, 2 表 2 统计分析表 Table 2 Statistical analysis table A B A+B C D C+D A+C B+D N=A+B+C+D A ; B, ; C, ; D 1) (p+)= A / (A+C) * 100% 2) (p-)= D / (B+D) * 100% 3) (relative accuracy)= (A+D) /N * 100% 4)(pf-)=(1- )* 100% 5)(pf+)=(1- )* 100% 2.4.3.2,, McNemar s (χ 2 ), [19] : 2 2 ( a - b 1) χ a b : a ; b 3 结果 3.1 SAT 法检验结果 2.4.3, 4 SAT, 3
2, : 685 表 3 Table 3 4 种致病菌 SAT 法与国标法结果统计表 The results of four pathogens tested with SAT and national standard SAT 92 42 134 0 196 196 92 238 330 15 8 23 0 217 217 15 225 240 17 2 19 0 41 41 17 43 60 2 0 2 0 28 28 2 28 30 3.2 方法性能指标及显著性差异分析, SAT, ( 4) 100%, 0%,,,,, (χ 2 =40.0) ; 100%, 0%, ; 100%,, 100% [19], ;, ;, ; SAT,,, 3.3 SAT 法测序方法评价分析 SAT, 42 30, 12 ; 8 7, 1 ; 2,, 5, 3 SAT + ( 6), 2.4.3, : 100%, 0%,, 90.4%( ), 94%(); 5.8%, 0.5%, [19],,, 7 4 讨论与结论 SAT Gen-probe (transcription mediated amplification, TMA), RNA, RNA,,,,,, Table 4 表 4 4 种致病菌 SAT 法与国标法比较性能指标及显著性差异结果 The performance indexes and the significant differences of four pathogens with SAT and national standard (%) (%) (%) (%) (%) χ 2 100 82.4 0 17.6 87.3 40.0 100 96.4 0 3.6 96.7 6.1 100 95.3 0 4.7 96.7 0.5 100 100 0 0 100 0
686 6 Table 5 表 5 不同检测项目测序结果情况 The results of difference of inspection item SAT 92 134 42 30 12 15 23 8 7 1 17 19 2 2 0 Table 6 表 6 几种致病菌 SAT 法与国标 + 测序法结果统计表 The results of several pathogens tested with SAT and national standard & sequencing SAT + 122 12 134 0 196 196 122 208 330 22 1 23 0 217 217 22 218 240 21 0 21 0 39 39 21 39 60 表 7 4 种致病菌测序后 SAT 法与国标法性比较性能指标及显著性差异结果 Table 7 The performance indexes and the significant difference of four pathogens with SAT by national standard and sequencing (%) (%) (%) (%) (%) χ 2 100 94.2 0 5.8 96.4 10.1 100 99.5 0 0.5 99.6 0 100 100 0 0 100 0 100 100 0 0 100 0, [12], SAT,,, 4 100%, ;, 42, 30, (94.2%), (5.8%), (96.4%); 8 7, SAT 99.5%, 0.5%, 99.6%; 2, 100%, SAT,
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