Uracil-DNA-glycosylase-assisted loop-mediated isothermal amplification for detection of bacteria from urine samples with reduced contamination

Zeng，Yingmin1; Liu，Meiling1; Xia，Yong1; Jiang，Xingyu2,3,4

2020-11-07

10.1039/d0an01001d

ANALYST   影响因子和分区

0003-2654

1364-5528

Loop-mediated isothermal amplification (LAMP) is a useful molecular biology technology for analytical applications, but it is prone to contamination because of escaped aerosols, leading to false positive results. This report establishes an integrated, rapid, and accurate method to detect bacteria in urine samples by incorporating uracil-DNA-glycosylase (UDG) into real-time loop-mediated isothermal amplification (RT-LAMP). To do this, nucleic acids from five clinically important uropathogens, including Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus mirabilis, and Enterococcus faecalis, were directly captured and concentrated using Flinders Technology Associates (FTA) Elute cards. Following elution, the extracted DNAs were specifically amplified with sets of LAMP primers. The added UDG in the modified LAMP reaction excises uracils from previously amplified products or contaminants and generates apyrimidinic (AP) sites, reducing false positive rates. This UDG-assisted RT LAMP strategy was able to degrade carryover contaminants to as little as 1 femtogram (10-15 g). The assay showed a limit of detection of 104 CFU mL-1 with a sensitivity of 94.1% and a specificity of 95.0%. Both the sensitivity and specificity were improved compared to LAMP carried out without UDG. Our results indicate that the UDG-assisted RT LAMP is of great potential for rapid and precise analysis of nucleic acids in real applications. This journal is

SCI ; EI

英语

其他

National key R & D Program of China[2018YFA0902600] ; National Natural Science Foundation of China[21535001][81730051][21761142006] ; Chinese Academy of Sciences[QYZDJ-SSW-SLH039][121D11KYSB20170026]

Chemistry

Chemistry, Analytical

WOS:000582413100029

ROYAL SOC CHEMISTRY

20204509454176

Isotherms ; Aromatic compounds ; Body fluids ; Contamination ; Escherichia coli ; Reaction rates

Biological Materials and Tissue Engineering:461.2 ; Biochemistry:801.2 ; Chemical Reactions:802.2 ; Organic Compounds:804.1

CHEMISTRY

2-s2.0-85094684040

Scopus

1.Department of Clinical Laboratory,Third Affiliated Hospital of Guangzhou Medical University,Guangzhou, Guangdong,No. 63 Duobao Road Liwan District,510150,China
2.Department of Biomedical Engineering,Southern University of Science and Technology,Shenzhen, Guangdong,No. 1088 Xueyuan Road Nanshan District,518055,China
3.Beijing Engineering Research Center for BioNanotechnology,CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety,CAS Center for Excellence in Nanoscience,National Center for NanoScience and Technology,Beijing,No. 11 Zhongguancun Beiyitiao,100190,China
4.University of Chinese Academy of Sciences,Beijing,19 A Yuquan Road, Shijingshan District,China

Zeng，Yingmin,Liu，Meiling,Xia，Yong,et al. Uracil-DNA-glycosylase-assisted loop-mediated isothermal amplification for detection of bacteria from urine samples with reduced contamination[J]. ANALYST,2020,145(21):7048-7055.

Zeng，Yingmin,Liu，Meiling,Xia，Yong,&Jiang，Xingyu.(2020).Uracil-DNA-glycosylase-assisted loop-mediated isothermal amplification for detection of bacteria from urine samples with reduced contamination.ANALYST,145(21),7048-7055.

Zeng，Yingmin,et al."Uracil-DNA-glycosylase-assisted loop-mediated isothermal amplification for detection of bacteria from urine samples with reduced contamination".ANALYST 145.21(2020):7048-7055.