Rapid detection of <i>Bacillus cereus</i> in rice by isothermal amplification with microfluidic chip method

doi:10.3785/j.issn.1008-9209.2020.06.101

Journal of Zhejiang University (Agriculture and Life Sciences)

2021, Vol. 47

Issue (2): 193-202 DOI: 10.3785/j.issn.1008-9209.2020.06.101

Food sciences

Rapid detection of Bacillus cereus in rice by isothermal amplification with microfluidic chip method

Zhen WANG¹(

),Lei HE²,Yingping XIAO³,Xiandong LU²,Yanhong LIU²,Wen LU¹,Shoufeng WANG⁴(

)

^1.Greentown Agricultural Testing Technology Co. , Ltd. , Hangzhou 310052, China
^2.Ningbo iGene Technology Co. , Ltd. , Ningbo 315000, Zhejiang, China
^3.Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
^4.Department of Basic Medicine, School of Medicine, Zhejiang University, Hangzhou 310058, China

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Abstract

Rapid detection of Bacillus cereus in rice by loop-mediated isothermal amplification (LAMP) with microfluidic chip method was established. Based on the published B. cereus hblA gene sequence, primers were designed, and the fluorescent dye SYTO-9 was added to the reaction system, which allowed to be tested in real-time fluorescence reading performed by LAMP with microfluidic chip. The specificity was verified by 24 strains, and the sensitivity, limit of detection (LOD) and repeatability were tested by positive plasmid and positive strain of B. cereus, respectively. The results showed that two strains of B. cereus were positive and 22 strains of non-B. cereus were negative. The sensitivity for B. cereus was 170 CFU/mL for pure cultures in 35 min and 10 μL^－1 for positive plasmids in 15 min. The sensitivity of this method was 10 times higher than that of the traditional method. The LOD for B. cereus inartificially contaminated rice was 570 CFU/g, and could be completed in 45 min. The detecting repeatability of the positive plasmid from B. cereus was good and its coefficient of variation (CV ) was 2.02%. It is indicated that LAMP with microfluidic chip method has high specificity, sensitivity and accuracy. It could be used for rapid detection of B. cereus in rice.

Key words： microfluidic chip loop-mediated isothermal amplification (LAMP) Bacillus cereus rice detection

Received: 10 June 2020 Published: 25 April 2021

CLC:

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Corresponding Authors: Shoufeng WANG E-mail: 705878750@ qq.com;sfwang@zju.edu.cn

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Cite this article:

Zhen WANG,Lei HE,Yingping XIAO,Xiandong LU,Yanhong LIU,Wen LU,Shoufeng WANG. Rapid detection of Bacillus cereus in rice by isothermal amplification with microfluidic chip method. Journal of Zhejiang University (Agriculture and Life Sciences), 2021, 47(2): 193-202.

URL:

http://www.zjujournals.com/agr/10.3785/j.issn.1008-9209.2020.06.101 OR http://www.zjujournals.com/agr/Y2021/V47/I2/193

微流控芯片恒温扩增技术快速检测米饭中的蜡样芽孢杆菌

为建立米饭中的蜡样芽孢杆菌微流控芯片恒温扩增快速检测方法，利用蜡样芽孢杆菌公开的hblA基因序列设计引物，加入荧光染料SYTO-9，采用微流控芯片通过环介导恒温扩增进行实时荧光读数，并通过24株菌株验证其特异性；分别用阳性质粒和阳性菌株测试其灵敏度、检出限和重复性。结果表明：2株蜡样芽孢杆菌呈阳性，22株非蜡样芽孢杆菌呈阴性；微流控芯片恒温扩增技术检测蜡样芽孢杆菌菌液的灵敏度为170 CFU/mL，检测时间在35 min内；使用合成的蜡样芽孢杆菌阳性质粒样品，其灵敏度为10 μL^－1，检测时间在15 min内，比传统分离鉴定方法的灵敏度高10倍；人工污染米饭中蜡样芽孢杆菌的检出限为570 CFU/g，并可在45 min内完成结果判定；蜡样芽孢杆菌阳性质粒检测重复性好，其变异系数（coefficient of variation, CV）为2.02%。综上所述，微流控芯片恒温扩增快速检测方法特异性好、灵敏度高、结果准确，可用于米饭类食品中蜡样芽孢杆菌的快速检测。

关键词： 微流控芯片, 环介导恒温扩增, 蜡样芽孢杆菌, 米饭, 检测

Table 1 Strains for experiment

Table 2 Primer sequences

Fig. 1 Temperature optimization results of LAMP reaction

Fig. 2 Specific testing results of LAMP with microfluidic chip for B. cereusIn figure A, 1: Bacillus cereus [CMCC (B) 63301]; 2: Bacillus thuringiensis (ATCC 10792); 3: Bacillus mycoides (ATCC 10206); 4: Bacillus subtilis [CMCC (B) 63501]; 5: Klebsiella peneumoniae (ATCC 4352); 6: Escherichia coli (ATCC 25922); 7: Enterohemorrhagic Escherichia coli O157∶H7 (CICC 21530); 8: Negative control. In figure B, 1: Bacillus cereus [CMCC (B) 63301]; 2: Salmonella typhimurium [CMCC (B) 50115]; 3: Enterobacter sakazakii (ATCC 29544); 4: Rhodococcus equi (ATCC 6939); 5: Vibrio parahaemolyticus (ATCC 17802); 6: Staphylococcus aureus (ATCC 6538); 7: Staphylococcus epidermidis [CMCC (B) 26069]; 8: Negative control. In figure C, 1: Bacillus cereus (LCLY 001); 2: Enterococcus faecalis (ATCC 29212); 3: Listeria monocytogenes (ATCC 19115); 4: Listeria ivanovii (ATCC 19119); 5: Pseudomonas aeruginosa (ATCC 27853); 6: Shigella flexneri [CMCC (B) 51572]; 7: β-hemolytic Streptococcus [CMCC (B) 32210]; 8: Negative control. In figure D, 1: Bacillus cereus [CMCC (B) 63301]; 2: Bacillus pumilus [CMCC (B) 63202]; 3: Bacillus licheniformis [CMCC (B) 63519]; 4: Bacillus amyloliquefaciens (ATCC 23845); 5: Bacillus megatherium (ATCC 35985); 6-8: Negative controls.

Fig. 3 Sensitivity testing results of LAMP with microfluidic chip for B. cereus1-5: The culture concentrations were 1.7×10⁵, 1.7×10⁴, 1.7×10³, 1.7×10², 1.7×10¹ CFU/mL, respectively; 6: Negative control.

Fig. 4 Sensitivity testing results of LAMP with microfluidic chip for pUC57-hblA1: Negative control; 2-8: Plasmid concentrations were 1×10⁰， 1×10¹， 1×10²， 1×10³， 1×10⁴， 1×10⁵， 1×10⁶ μL^－1， respectively.

Fig. 5 Repeatability testing results of LAMP with microfluidic chip for pUC57-hblA1-8: Results of eight repeated tests of 1×10⁴ μL^－1 plasmid.

Table 3 Analysis of the coefficient of variation on the detection of B. cereus by LAMP with microfluidic chip

Fig. 6 Limit of detection of B.cereus in artificially polluted rice by LAMP with microfluidic chip1-7: The culture concentrations of B. cereus in rice samples were 5.7×10⁶, 5.7×10⁵, 5.7×10⁴, 5.7×10³, 5.7×10², 5.7×10¹, 5.7×10⁰ CFU/mL, respectively; 8: Negative control.

Fig. 7 Microfluidic chip


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