幽门螺杆菌ureC和23S rDNA数字PCR检测体系的建立

doi:10.19586/j.2095-2341.2024.0012

生物技术进展 ›› 2024, Vol. 14 ›› Issue (5): 868-874.DOI: 10.19586/j.2095-2341.2024.0012

• 研究论文 • 上一篇

幽门螺杆菌ureC和23S rDNA数字PCR检测体系的建立

范宏博¹^,²(), 胡良勇², 胡松青¹()

^1.华南理工大学食品科学与工程学院，广州 510630
^2.广州计量检测技术研究院，广州 510700

收稿日期:2024-01-18 接受日期:2024-07-02 出版日期:2024-09-25 发布日期:2024-10-22
通讯作者: 胡松青
作者简介:范宏博 E-mail：422488792@qq.com；
基金资助:
广东省重点领域研发计划项目(2020B0404010002)

Establishment of Digital PCR Detection System for Helicobacter pyloriureC and 23S rDNA

Hongbo FAN¹^,²(), Liangyong HU², Songqing HU¹()

^1.Department of Science and Technology Development，South China University of Technology，Guangzhou 510630，China
^2.Guangzhou Institute of Measurement and Testing Technology，Guangzhou 510700，China

Received:2024-01-18 Accepted:2024-07-02 Online:2024-09-25 Published:2024-10-22
Contact: Songqing HU

摘要/Abstract

摘要：

为了建立幽门螺杆菌（Helicobacter pylori，Hp）ureC和23S rDNA基因的数字PCR检测体系，为Hp检测提供一个可靠的参考方法，根据Hp ureC和23S rDNA基因序列，设计了特异性引物和探针，采用质控菌评估了检测特异性；通过设定引物浓度和退火温度的梯度，对检测参数进行了优化；通过梯度法稀释DNA模板，评估了检测灵敏度；使用不同浓度模板开展了重复性检测，以评估检测精密度。经过评估，该检测方法能够特异性地用于ureC和23S rDNA基因的检测，并且不受大肠杆菌等细菌干扰。ureC和23S rDNA的最佳反应温度均为55.8 ℃，而最佳引物浓度分别为550和650 nmol·L^-1，通过线性分析，两种基因的拟合度R² 值分别高达0.999 1和0.999 7，显示了良好的线性关系。此外，不同浓度样品的变异系数（CV）均小于10%，说明该检测方法具有高度的重复性。研究成功建立了Hp ureC和23S rDNA基因数字PCR检测体系，具备高度的特异性、灵敏度和重复性，为幽门螺杆菌的检测、诊断和科学研究提供了一个可靠的检测方法和技术支持。

关键词: 幽门螺杆菌, ureC, 23S rDNA, 数字PCR

Abstract:

This study aimed to establish the digital PCR system for detection of Helicobacter pylori （Hp） genes ureC and 23S rDNA， providing an reliable reference detection method for Hp diagnosis. Specific primers and probes were designed based on the Hp ureC and 23S rDNA sequences， and quality control bacteria was used to conduct specificity testing. The gradient method was used to set the primer concentrations and annealing temperatures to optimize the reaction conditions. The sensitivity was detected using gradient diluted DNA as templates. The accuracy was detected using different concentration templates with multiple repeated tests. The designed primers and probes could specifically detect Hp ureC and 23S rDNA without the interference from E. coli and other bacteria. The optimal reaction temperature for ureC and 23S rDNA was both 55.0 ℃， the optimal primer concentration was 550 and 650 nmol·L^-1， the linear fitting degree R² was 0.999 1 and 0.999 7， and the CV values of samples with different concentrations was less than 10%. The Hp digital PCR detection system for ureC and 23S rDNA established in this study has the advantages of high specificity， sensitivity， and repeatability， which can provide accurate and reliable technical support for Hp pathological detection and scientific research.

Key words: Helicobacter pylori, ureC, 23S rDNA, digital PCR

中图分类号:

Q789

范宏博, 胡良勇, 胡松青. 幽门螺杆菌ureC和23S rDNA数字PCR检测体系的建立[J]. 生物技术进展, 2024, 14(5): 868-874.

Hongbo FAN, Liangyong HU, Songqing HU. Establishment of Digital PCR Detection System for Helicobacter pyloriureC and 23S rDNA[J]. Current Biotechnology, 2024, 14(5): 868-874.

图/表 6

图1 引物和探针检验A: ureC基因检测散点图；B：ureC基因直方图；C：23S rDNA基因检测散点图；D：23S rDNA基因直方图

Fig. 1 Detection of primers and probes

图2 检测特异性评估A：ureC检测特异性；B：23S rDNA检测特异性。1—Hp，2—空白，3—金黄色葡萄球菌，4—唾液链球菌，5—嗜酸乳杆菌，6—大肠杆菌。

Fig. 2 Testing of the specificity

图3 退火温度优化A: ureC；B: 23S rDNA

Fig. 3 Optimization of annealing temperature

图4 引物浓度优化A: ureC；B :23S rDNA

Fig. 4 Optimization of primer concentration

图5 灵敏度检验A: ureC数字PCR散点图；B： ureC线性回归分析图；C： 23S rDNA数字PCR散点图；D： 23S rDNA线性回归分析图

Fig. 5 Testing of sensitivity

表1 精密度评估

Table 1 Testing of precision

		1	2	3	4	5	6	平均浓度/（copies·μL^-1）	标准差（SD）	变异系数（CV/%）
ureC	样品1	474.0	495.0	481.0	506.0	513.0	482.0	491.8	15.4	3.1
	样品2	77.4	75.2	81.2	75.6	82.1	82.1	78.9	3.2	4.1
	样品3	7.5	6.8	7.9	7.4	7.7	6.6	7.3	0.5	7.1
23S rDNA	样品1	415.0	426.2	381.7	394.2	410.5	398.0	404.3	16.0	4.0
	样品2	20.8	22.3	23.4	23.9	24.1	21.0	22.7	1.5	6.6
	样品3	8.7	9.3	9.1	9.6	7.7	8.9	8.9	0.7	7.4

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幽门螺杆菌ureC和23S rDNA数字PCR检测体系的建立

Establishment of Digital PCR Detection System for Helicobacter pyloriureC and 23S rDNA

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