Development of Fluorescence Quantitative Lyophilized Detection Kit Based on Escherichia coli O157∶H7

doi:10.13560/j.cnki.biotech.bull.1985.2021-0768

Abstract

Abstract:

Escherichia coli O157∶H7,as a food-borne pathogen,is widely distributed and causes great harms. To develop a kit based on E. coli O157∶H7 that can not only achieve rapid,simple and sensitive detection,but also achieve room temperature storage and transportation,this study utilized real-time fluorescence quantitative polymerase chain reaction（qPCR）,combinig with vacuum lyophilizing technology,and developed an E. coli O157∶H7 fluorescence quantitative lyophilizing detection kit. This kit retains nucleic acid detection performance,and is easy to store and transport at room temperature,and reduces aerosol pollution. The developed kit adopts lyophilizing technology to retain the detection performance of nucleic acid amplification reagents. After rehydration,the accumulation of fluorescent signals is monitored in real time through cyclic amplification under the action of Taq DNA polymerase to achieve quantitative fluorescence detection. By the proposed method 2.1 copies/μL of eaeA gene can be detected within 40 min. This technology provides a fine research foundation and technical reference for the detection of E. coli O157∶H7,and fills the lack of detection kits with high sensitivity and easy storage in the market.

Key words: E. coli O157∶H7, nucleic acid, fluorescent quantitative PCR, lyophilized protective agent, kits

SU Yuan, ZHU Long-jiao, CAO Ji-juan, LIU Jian-long, XU Wen-tao. Development of Fluorescence Quantitative Lyophilized Detection Kit Based on Escherichia coli O157∶H7[J]. Biotechnology Bulletin, 2022, 38(3): 264-275.

Figures/Tables 14

Table 1 Classification of the lyoprotectant[16]

分类 Classification	冻干保护剂名称 Name of lyoprotectant	功能 Function
糖类	海藻糖、乳糖、蔗糖、果糖、麦芽糖	稳定剂
氨基酸类	精氨酸、甘氨酸、色氨酸	缓冲剂
盐类	磷酸盐（PBS）、醋酸盐、柠檬酸盐和琥珀酸盐	缓冲剂
聚合物类	聚乙烯吡咯烷酮（PVP）、聚乙二醇	增强效果
糖脂类	牛血清白蛋白（BSA）	增强效果
多元醇类	甘露醇、甘油、山梨醇	填充剂

Table 2 Primers' sequences of qPCR

引物名称 Primer name	引物序列 Primer sequence（5'-3'）
16S rDNA-F	CAACGCGAAGAACCTTACC
16S rDNA-R	CGACAGCCATGCANCACCT
E. coli O157:H7-F	CGTCATGGTACGGGTAATGAA
E. coli O157:H7-R	TAACGGCTGCCCGATAATG
E. coli O157:H7-T	FAM-TGGTCTCAGCAAATCGAGCCACA-TAMRA

Fig. 1 Agarose gel electrophoresis image A：E. coli O157:H7 genome. M：2 000 bp DNA ladder. 1-3：E. coli O157:H7 template. B：16S rDNA PCR product. M：2 000 bp DNA ladder. 1-3：90 bp target fragment of E. coli O157:H7

Fig. 2 Concentration optimization of F and R of E. coli O157：H7 A：Quantitative graph. B：Electrophoresis graph. C：Bar graph. M：DNA marker Ι;1-3：0.2 μmol/L F and R,negative control,and control check;4-6：0.3 μmol/L F and R,negative control,and control check;7-9：0.4 μmol/L F and R,negative control,and control check;10-12：0.5 μmol/L F and R,negative control,and control check;13-15：0.6 μmol/L F and R,negative control,and control check

Fig. 3 Optimization of the ratios of primers to probes of E. coli O157:H7 A：Quantitative graph. B：Electrophoresis graph. C：Bar graph. M：DNA marker I;1-3：primer:probe is 3:1,negative control,and control check;4-6：primer:probe is 2:1,negative control,and control check;7-9：primer:probe is 1:1,negative control,and control check;10-12：primer:probe is 1:2,negative control,and control check;13-15：primer:probe is 1:3,negative control,and control check

Fig. 4 Optimization of annealing temperature for E. coli O157:H7 A：Quantitative graph. B：Electrophoresis graph. C：Bar graph. M：DNA marker Ι;1-3：50℃,negative control,and control check;4-6：53℃,negative control,control check;7-9：56℃,control check;10-12：59℃,negative control,and control check;13-15：62℃,negative control,and control check;16-18：65℃,negative control,and control check

Fig. 5 Sensitivity of the established E. coli O157：H7 detection method A：Quantitative graph of the sensitivity of the detection method. B：The linear relationship between the Ct and the copies of E. coli O157：H7

Fig. 6 Specificity of the established E. coli O157:H7 detection method

Fig. 7 Effect of lyoprotectant on qPCR amplification

Fig. 8 Freeze-drying protective effect of lyoprotectant on qPCR system

Fig. 9 Establishment of the fluorescent quantitative lyoph-ilized detection kit A：Detection effect. B：Morphology of lyophilized samples

Fig. 10 Reproducibility of the fluorescence quantitative lyophilized detection kit A：Amplification curve of the intra-batch kit. B：Amplification curve of the inter-batch kit

Fig. 11 Detection effect of the fluorescence quantitative lyophilized detection kit

Fig. 12 Detection of actual samples A：Fluorescence quantitative lyophilized detection kit. B：National standard method

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