The Study of a Novel Nucleic Acid Detection Technology by Double-stranded Probe Real-time PCR

doi:10.13523/j.cb.2008124

China Biotechnology

2020, Vol. 40

Issue (11): 28-34 DOI: 10.13523/j.cb.2008124

The Study of a Novel Nucleic Acid Detection Technology by Double-stranded Probe Real-time PCR

LIU Li-yan^1**,LIU Qi-qi^1**,ZHANG Ying²,WANG Sheng-qi^1***(

)

1 Institute of Radiation Medicine, Academy of Military Medical Science, Beijing 100850, China
2 Zhengzhou Maylink Biotechnology Co. Ltd, Zhengzhou 450000, China

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Abstract

Objective: Using a “double-stranded probe” real-time fluorescent PCR technology to improve the sensitivity of HBV nucleic acid detection, complete the genotype detection of metabolic enzyme CYP2C19 ^*2 in a tube. Methods: The double-stranded probe and the TaqMan probe was used to simultaneously detect different concentrations of HBV in serum samples by Shanghai Hongshi SLAN 96 real-time fluorescent PCR instrument. Then, according to the Ct value of nucleic acid detection by instrument to statistical analysis of results; the double-stranded probe was used to detect samples of different genotypes of metabolic enzyme CYP2C19^*2 in a tube, and the detection of nucleic acid Ct value and genotype analysis were performed by Shanghai Hongshi SLAN 96 real-time fluorescent PCR instrument. Results: In the detection of HBV serum samples at different concentrations, the fluorescence background of the double-stranded probe was low and the detection sensitivity was higher than the TaqMan probe. And significant differences were noted between the two probes (P<0.05);The metabolic enzyme CYP2C19^*2 genotypes of 36 samples were detected using the double-stranded probe, the results were consistent with those of Sanger sequencing. Conclusion: The double-stranded probe real-time fluorescent PCR detection technology can complete the highly sensitive nucleic acid detection of the target gene and also the genotype analysis.

Key words： Double-stranded probe TaqMan probe Real-time PCR HBV CYP2C19^*2

Received: 15 August 2020 Published: 11 December 2020

ZTFLH:

Q819

Corresponding Authors: Sheng-qi WANG E-mail: sqwang@bmi.ac.cn

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	Li-yan LIU
	Qi-qi LIU
	Ying ZHANG
	Sheng-qi WANG

Cite this article:

LIU Li-yan,LIU Qi-qi,ZHANG Ying,WANG Sheng-qi. The Study of a Novel Nucleic Acid Detection Technology by Double-stranded Probe Real-time PCR. China Biotechnology, 2020, 40(11): 28-34.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2008124 OR https://manu60.magtech.com.cn/biotech/Y2020/V40/I11/28

Fig.1 Structure of double-stranded probe

Fig.2 Detection values of the fluorescent background using double-stranded probe and TaqMan probe The “1” group straight line shows the detection value of the fluorescent background signal of the TaqMan probe. The “2” group straight line shows the detection value of the fluorescent background signal of the double-stranded probe

Table 1 Primer and probe sequences of HBV, and CYP2C19^*2

Table 2 The Ct values of 24 quantitative HBV samples by double-stranded probe and TaqMan probes detection

Table 3 Detection results of three kinds of CYP2C19^*2 genotypes by double-stranded probe

Fig.3 The detection of 36 samples of different CYP2C19^* 2 genotypes by double-stranded probe (a) 12 samples of mutation genotype (A/A) by double-stranded probe detection (b) 12 samples of wild genotype (G/G) by double-stranded probe detection (c) 12 samples of heterozygous genotype(G/A) by double-stranded probe detection


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