Elimination of the endogenous nucleic acid in the real-time quantitative polymerase chain reaction by restriction endonuclease digestion_West China Medical Journal

Authors：

LIU Jiangang ¹ , WANG Qiujing ¹ , LIU Minggang ² , SONG Zhao ¹ ,  CHEN Qian ²

1. Department of Neurosurgery, Shenzhen Hospital of Southern Medical University, Shenzhen, Guangdong 518000, P. R. China;
2. Department of Neurosurgery, Shenzhen Children’s Hospital, Shenzhen, Guangdong 518026, P. R. China;

Corresponding author：

CHEN Qian, Email: chenqian68@126.com

Keywords：

Intracranial infection; Real-time quantitative polymerase chain reaction; Endogenous nucleic acid pollution

DOI：

10.7507/1002-0179.201803109

Video：

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Abstract Full text Figures/Tables Video References Cited by

ObjectiveTo establish a method that can eliminate the pollution of endogenous nucleic acid in the real-time quantitative polymerase chain reaction (PCR) reaction system, which can be used to reduce or eliminate the false positive rate of real-time PCR assay in detection of postoperative intracranial bacteria infection.MethodsAt first, eliminated the pollution of endogenous nucleic acid in the real-time PCR reaction system. Then, with mixed bacteria DNA as a template, multiple PCR was used to specifically identify the gram-negative bacteria. Meanwhile, evaluated the text line and sensitivity of the multiple PCR after eliminating pollution in detecting the DNA of the mixed bacteria.ResultsThe method established could quickly eliminate the pollution of endogenous nucleic acid in the real-time PCR reaction system, and it didn’t affect the Taq enzyme activity and the amplification efficiency in PCR system, with the minimum detection limit of 10² CFU/mL (Staphylococcus aureus and Pseudomonas aeruginosa), which was the same to the culture method. The enzyme cutting method had no significant effect on the activity and amplification efficiency of the enzyme in PCR system, It had no effect on PCR reaction system and primer specificity (Ct=32, ΔRn=200). However, the filtration method significantly reduced the PCR amplification efficiency (Ct=32, ΔRn=150).ConclusionsThis method can easily and rapidly eliminate the pollution of endogenous nucleic acid in the real-time PCR reaction system, and greatly reduce the false positive of PCR detection. It is able to timely and accurately diagnose the intracranial bacteria infection, which is significant for clinical testing.

Citation： LIU Jiangang, WANG Qiujing, LIU Minggang, SONG Zhao, CHEN Qian. Elimination of the endogenous nucleic acid in the real-time quantitative polymerase chain reaction by restriction endonuclease digestion. West China Medical Journal, 2018, 33(6): 744-747. doi: 10.7507/1002-0179.201803109 Copy

1.	时红云, 鲁燕飞, 叶菊花. 神经外科术后患者颅内感染情况及抗生素的使用. 中国实用神经疾病杂志, 2015, 18(11): 91-92.
2.	Saramma PP, Krishnakumar K, Sarma PS. Alcohol-based hand rub and surgical site infection after elective neurosurgery: an intervention. Neurol India, 2011, 59(1): 12-17.
3.	徐廷伟, 敖祥生, 秦永芳, 等. 神经外科患者术后颅内感染危险因素分析. 中华医院感染学杂志, 2016, 26(8): 1758-1760.
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5.	崔向丽, 林松, 隋大立, 等. 神经外科术后颅内感染的诊疗进展. 中华神经外科杂志, 2014, 30(3): 312-314.
6.	陈松劲, 吴晓燕, 倪侃翔. 878 份脑脊液标本培养的病原菌及耐药性分析. 中华医院感染学杂志, 2008, 18(10): 1460-1462.
7.	巴华君, 孙军. 开颅手术后脑脊液培养病原菌分布及耐药性分析. 中华医院感染学杂志, 2012, 22(2): 426-428.
8.	Corless CE, Guiver M, Borrow R, et al. Contamination and sensitivity issues with a real-time Universal 16S rRNA PCR. J Clin Microbiol, 2000, 38(5): 1747-1752.
9.	Lupan I, Ianc MB, Ochiş C, et al. The evidence of contaminant bacterial DNA in severa l commercial Taq polymerases. Romanian biotechnological letters, 2013, 18(1): 8007-8012.
10.	高路, 何聪芬, 李萌, 等. 金黄色葡萄球菌快速检测技术的研究进展. 食品科学技术学报, 2014, 32(2): 51-55.
11.	马群飞, 林坚. GB/T8538-2008 饮用天然矿泉水检验方法存在的微生物学问题. 中国卫生标准管理, 2011, 2(4): 44-47.
12.	Hilali F, Saulnier P, Chachaty E, et al. Decontamination of polymerase chain reaction reagents for detection of low concentrations of 16S rRNA genes. Mol Biotechnol, 1997, 7(3): 207-216.
13.	Carroll NM, Adamson P, Okhravi N. Elimination of bacterial DNA from Taq DNA polymerases by restriction endonuclease digestion. J Clin Microbiol, 1999, 37(10): 3402-3404.
14.	Khater WS, Elabd SH. Identification of common bacterial pathogens causing meningitis in culture-negative cerebrospinal fluid samples using real-time polymerase chain reaction. Int J Microbiol, 2016: 4197187.

1. 时红云, 鲁燕飞, 叶菊花. 神经外科术后患者颅内感染情况及抗生素的使用. 中国实用神经疾病杂志, 2015, 18(11): 91-92.
2. Saramma PP, Krishnakumar K, Sarma PS. Alcohol-based hand rub and surgical site infection after elective neurosurgery: an intervention. Neurol India, 2011, 59(1): 12-17.
3. 徐廷伟, 敖祥生, 秦永芳, 等. 神经外科患者术后颅内感染危险因素分析. 中华医院感染学杂志, 2016, 26(8): 1758-1760.
4. Brouwer MC, Tunkel AR, van de Beek D. Epidemiology, diagnosis, and antimicrobial treatment of acute bacterial meningitis. Clin Microbiol Rev, 2010, 23(3): 467-492.
5. 崔向丽, 林松, 隋大立, 等. 神经外科术后颅内感染的诊疗进展. 中华神经外科杂志, 2014, 30(3): 312-314.
6. 陈松劲, 吴晓燕, 倪侃翔. 878 份脑脊液标本培养的病原菌及耐药性分析. 中华医院感染学杂志, 2008, 18(10): 1460-1462.
7. 巴华君, 孙军. 开颅手术后脑脊液培养病原菌分布及耐药性分析. 中华医院感染学杂志, 2012, 22(2): 426-428.
8. Corless CE, Guiver M, Borrow R, et al. Contamination and sensitivity issues with a real-time Universal 16S rRNA PCR. J Clin Microbiol, 2000, 38(5): 1747-1752.
9. Lupan I, Ianc MB, Ochiş C, et al. The evidence of contaminant bacterial DNA in severa l commercial Taq polymerases. Romanian biotechnological letters, 2013, 18(1): 8007-8012.
10. 高路, 何聪芬, 李萌, 等. 金黄色葡萄球菌快速检测技术的研究进展. 食品科学技术学报, 2014, 32(2): 51-55.
11. 马群飞, 林坚. GB/T8538-2008 饮用天然矿泉水检验方法存在的微生物学问题. 中国卫生标准管理, 2011, 2(4): 44-47.
12. Hilali F, Saulnier P, Chachaty E, et al. Decontamination of polymerase chain reaction reagents for detection of low concentrations of 16S rRNA genes. Mol Biotechnol, 1997, 7(3): 207-216.
13. Carroll NM, Adamson P, Okhravi N. Elimination of bacterial DNA from Taq DNA polymerases by restriction endonuclease digestion. J Clin Microbiol, 1999, 37(10): 3402-3404.
14. Khater WS, Elabd SH. Identification of common bacterial pathogens causing meningitis in culture-negative cerebrospinal fluid samples using real-time polymerase chain reaction. Int J Microbiol, 2016: 4197187.

West China Medical Journal

Elimination of the endogenous nucleic acid in the real-time quantitative polymerase chain reaction by restriction endonuclease digestion

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