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：

Export PDF Favorites Scan Get Citation

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.	時紅云, 魯燕飛, 葉菊花. 神經外科術后患者顱內感染情況及抗生素的使用. 中國實用神經疾病雜志, 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.	徐廷偉, 敖祥生, 秦永芳, 等. 神經外科患者術后顱內感染危險因素分析. 中華醫院感染學雜志, 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.	崔向麗, 林松, 隋大立, 等. 神經外科術后顱內感染的診療進展. 中華神經外科雜志, 2014, 30(3): 312-314.
6.	陳松勁, 吳曉燕, 倪侃翔. 878 份腦脊液標本培養的病原菌及耐藥性分析. 中華醫院感染學雜志, 2008, 18(10): 1460-1462.
7.	巴華君, 孫軍. 開顱手術后腦脊液培養病原菌分布及耐藥性分析. 中華醫院感染學雜志, 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, Ochi? 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, 何聰芬, 李萌, 等. 金黃色葡萄球菌快速檢測技術的研究進展. 食品科學技術學報, 2014, 32(2): 51-55.
11.	馬群飛, 林堅. GB/T8538-2008 飲用天然礦泉水檢驗方法存在的微生物學問題. 中國衛生標準管理, 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. 時紅云, 魯燕飛, 葉菊花. 神經外科術后患者顱內感染情況及抗生素的使用. 中國實用神經疾病雜志, 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. 徐廷偉, 敖祥生, 秦永芳, 等. 神經外科患者術后顱內感染危險因素分析. 中華醫院感染學雜志, 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. 崔向麗, 林松, 隋大立, 等. 神經外科術后顱內感染的診療進展. 中華神經外科雜志, 2014, 30(3): 312-314.
6. 陳松勁, 吳曉燕, 倪侃翔. 878 份腦脊液標本培養的病原菌及耐藥性分析. 中華醫院感染學雜志, 2008, 18(10): 1460-1462.
7. 巴華君, 孫軍. 開顱手術后腦脊液培養病原菌分布及耐藥性分析. 中華醫院感染學雜志, 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, Ochi? 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, 何聰芬, 李萌, 等. 金黃色葡萄球菌快速檢測技術的研究進展. 食品科學技術學報, 2014, 32(2): 51-55.
11. 馬群飛, 林堅. GB/T8538-2008 飲用天然礦泉水檢驗方法存在的微生物學問題. 中國衛生標準管理, 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

Abstract Full text Figures/Tables Video References Cited by

Previous Article

Next Article

Format

Content