Development of a Innovative Fluorescent Quantitative PCR Method for Salmonella Based on Fluorescent Self-quenching Primers

doi:10.13560/j.cnki.biotech.bull.1985.2021-0101

Abstract

Abstract:

Salmonella is a common food-borne pathogen,and food-borne disease caused by Salmonella is in the top of a list. Fluorescence quantitative polymerase chain reaction（FQ-PCR）is an accurate and reliable amplification technique for nucleic acid quantification. The commonly used fluorescent-labeled probe methods make the amplification systems complicated and the cost of primer synthesis high. In order to simplify the FQ-PCR amplification system and reduce the modification of the labeling group,it is planned to conduct an innovative FQ-PCR（IFQ-PCR）for the quantitative detection of template DNA through single-labeled fluorescent self-quenching primers. The single-labeled hairpin type fluorescent self-quenching primer was designed according to Salmonella-specific genes,and applied to FQ-PCR to conduct the detection of Salmonella. The designed self-quenching primer was integrated with probe,only a single label was required,and the amplification system did not require the additional probes or dyes,thus the amplification system was simplified and detection cost was reduced. Meanwhile,the hairpin structure of the primers improved the specificity of the system. At the optimal primer concentration（0.4μmol/L）,the logarithm of the Salmonella concentration showed a good linear relationship with cycle threshold value in the concentration range of 10¹-10⁵ CFU/mL,with R² as high as 0.99 and detection limit as low as 2 CFU/ mL,the amplification detection was completed within 1.5 h,and the stability of the method met the requirements. Therefore,an IFQ-PCR based on fluorescent self-quenching primers was developed to have the simple,rapid,sensitive,specific and low-cost detection of Salmonella.

Key words: fluorescence quantitative PCR, fluorescence self-quenching primers, Salmonella, quantitative detection

FENG Min, LI Shu-ting, ZHANG Yang-zi, SU Yuan, ZHU Long-jiao, CAO Ji-juan, LIU Hai-yan, XU Wen-tao. Development of a Innovative Fluorescent Quantitative PCR Method for Salmonella Based on Fluorescent Self-quenching Primers[J]. Biotechnology Bulletin, 2021, 37(11): 285-292.

Figures/Tables 10

Table 1 Primer and target gene sequence

名称Name	序列Sequence（5'-3'）	碱基数Number of bases/nt
上游引物Forward primer（FP）	CGGGTCAAGGCTGAGGAA	18
原始下游引物Original reverse primer（ORP）	TGCTGAAGTTGAGGATGTTATTCG	24
下游引物Reverse primer（RP）靶基因Target gene	CGAATATGCTGAAGTTGAGGATGTTATT（FAM）CG TGCTGAAGTTGAGGATGTTATTCGCAAAGGGATCCGTCA GACCTCTGGCAGTACCTTCCTCAGCCTTGACCCG	30 85

Fig.1 Schematic diagram

Fig.2 Primer concentration optimization A: CT value comparison chart. B: Amplification curve. C: Agarose gel electrophoresis chart. Lanes 1-5 are amplified product at primer concentrations of 500 nmol/L, 400 nmol/L, 300 nmol/L, 200 nmol/L, 100 nmol/L, respectively

Fig.3 Sensitivity detection A: Amplification curve: the Salmonella concentrations corresponding to the big red line, black line, green line, orange line, purple line and pink line are 105 CFU/mL, 104 CFU/mL、103 CFU/mL, 102 CFU/mL, 101 CFU/mL and 100 CFU/mL (the attached shows the relationship between the CT value corresponding to the amplification curve and lgCsalmonella); B: agarose gel electrophoresis diagram: the concentration of Salmonella corresponding to lanes 1-6 is 105 CFU/mL, 104 CFU/mL, 103 CFU/mL, 102 CFU/mL, 101 CFU/mL and 100 CFU/mL

Fig.4 Standard curve

Fig.5 Specific detection A: Amplification curve: The food-borne pathogen corresponding to the big red line is Salmonella, and the other color lines represent Staphylococcus aureus, Bacillus cereus, Escherichia coli, Listeria monocytogenes, and Vibrio parahaemolyticus. B :Agarose gel electrophoresis diagram: lanes 1-6 are the products of DNA amplification of Salmonella, Staphylococcus aureus, Bacillus cereus, Escherichia coli, Listeria monocytogenes, and Vibrio parahaemolyticus, respectively

Fig. 6 Test results of actual samples

Table 2 IFQ-PCR method for the detection results of different concentrations of Salmonella in milk samples

沙门氏菌的浓度 Concentration of Salmonella/（CFU·mL^-1）	CT值 CT value			±S	RSD
10⁵	16.9	16.98	16.89	16.9±0.05	0.29%
10⁴	20.26	20.29	20.18	20.2±0.05	0.28%
10³	23.66	23.63	23.55	23.6±0.06	0.24%
10²	26.49	26.43	26.34	26.4±0.08	0.29%

Fig.7 Sensitivity detection of FQ-PCR based on dye method A: Amplification curve: The Salmonella concentrations corresponding to the blue line, orange line, purple line, yellow line, gray line, and green line are 105 CFU/mL, 104 CFU/mL, 103 CFU/mL, 102 CFU/mL, 101 CFU/mL and 100 CFU/mL (The attached shows the relationship between the CT value corresponding to the amplification curve and lgCsalmonella). B: FQ-PCR method standard curve

Table 3 Comparison of IFQ-PCR method and FQ-PCR method [13-16, 19-23]

方法Method	优点Advantage	缺点Disadvantage
IFQ-PCR法 IFQ-PCR method	引物的修饰简单、合成成本相对较低、扩增体系简单、可减少引物二聚体和碱基错配的发生	引物的设计要求高
FQ-PCR染料法 FQ-PCR dye method	无需标记、灵敏度高、成本低、可通过熔解曲线分析结果	特异性差、扩增体系需额外添加染料
探针法 Probe method	特异性最强、不受非特异性扩增与引物二聚体的影响	探针的设计复杂、标记成本高、扩增体系复杂

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