Establishment of loop‑mediated isothermal amplification for rapid detection of Pseudomonas aeruginosa

Li, Chao; Shi, Yaoqiang; Yang, Guangying; Xia, Xueshan; Mao, Xiaoqin; Fang, Yue; Zhang, A‐Mei; Song, Yuzhu

doi:10.3892/etm.2018.6910

Cited by 8 publications

(8 citation statements)

References 20 publications

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“…A total of 173 genome sequences of Mycobacterium were downloaded and analyzed. A specific MTB gene was selected using the method from our previous study 10 . Briefly, 173 genomes of Mycobacterium (including 10 MTB sequences) were downloaded from the NCBI to a local database and blasted using BLAST software.…”

Section: Methodsmentioning

confidence: 99%

Detection of Mycobacterium tuberculosis in clinical sputum by a unique gene in MTB strains called Conserved protein TB18.5 (TB18.5)

Luo

Song

et al. 2021

Clinical Laboratory Analysis

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A rapid and accurate diagnosis increases the treatment effect and decreases the mortality of tuberculosis (TB) patients. The purpose of this study was to establish an accurate, unique, and rapid molecular diagnostic technique to screen Mycobacterium tuberculosis (MTB) from clinical sputum. A unique gene in MTB strains called conserved protein TB18.5 (TB18.5) was selected by bioinformatics analysis. Two pairs of primers were designed to amplify TB18.5 using the nested polymerase chain reaction (PCR) or quantitative real‐time PCR. Nine pathogens and the MTB strain were used to determine the specificity of the TB18.5 gene. The sensitivity assay was performed after optimizing the PCR conditions. The correct fragment was amplified when a 10 copy number template was used. A total of 232 sputum samples were collected from TB patients (from 2019 to 2020) to evaluate the accuracy of the molecular method in this study. MTB was first detected using the BACTEC MGIT‐960 culture test and the Gene Xpert MTB/RIF assay. Totals of 195 (84.05%), 182 (78.45%), and 162 (69.83%) sputum samples were determined to be infected with MTB using nested PCR, the Gene Xpert MTB/RIF assay, and the BACTEC MGIT‐960 culture test, respectively. In summary, a rapid, unique, and sensitive molecular method was established to diagnose TB infection in clinical sputum samples.

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Section: Methodsmentioning

confidence: 99%

Detection of Mycobacterium tuberculosis in clinical sputum by a unique gene in MTB strains called Conserved protein TB18.5 (TB18.5)

Luo

Song

et al. 2021

Clinical Laboratory Analysis

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“…We initially screened for genes speci c to S. aureus by local BLAST and online BLAST was used to further identify the target gene, according to previously published procedures (Li et al 2019). LAMP primers were designed using Primer Explorer V5 (https://primerexplorer.jp/lampv5/index.html) based on the target sequence.…”

Section: Screening Of Target Genes and Primer Designmentioning

confidence: 99%

Rapid Visualization and Detection of Staphylococcus Aureus Based on Loop-Mediated Isothermal Amplification

Zeng

2021

Preprint

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Staphylococcus aureus is a common clinical bacterial pathogen that can cause a diverse range of infections. The establishment of a rapid and reliable assay for the early diagnosis and detection of S. aureus is of great significance. In this study, we developed a closed-tube loop-mediated isothermal amplification (LAMP) assay for the visual detection of S. aureus using the colorimetric indicator hydroxy naphthol blue (HNB). The LAMP reaction was optimized by adjusting the amplification temperature, the concentrations of Mg2+, dNTP, and HNB, and the incubation time. In the optimized reaction system, the specificity of LAMP for S. aureus was 100%. The results established that this method accurately identified S. aureus, with no cross-reactivity with 16 non-S. aureus strains. The limit of detection (LOD) of LAMP was 8 copies/reaction of purified plasmid DNA or 400 colony-forming units/reaction of S. aureus. Compared with conventional PCR, LAMP lowered the LOD by 10-fold. Finally, 220 clinically isolated strains of S. aureus and 149 non-S. aureus strains were used to evaluate the diagnostic efficacy of LAMP. The findings indicated that LAMP is a reliable test for S. aureus and could be a promising tool for the rapid diagnosis of S. aureus infections.

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“…The positive plasmids with resistance genes were constructed as described in a previous study (Li et al, 2019). In brief, the DNA fragments of the target genes were obtained by PCR reactions using the genomic DNA of E. coli as a template.…”

Section: Construction and Verification Of Positive Plasmidsmentioning

confidence: 99%

A rapid and accurate method for the detection of four aminoglycoside modifying enzyme drug resistance gene in clinical strains of Escherichia coli by a multiplex polymerase chain reaction

Shi

Yang

et al. 2020

PeerJ

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Background Antibiotics are highly effective drugs used in the treatment of infectious diseases. Aminoglycoside antibiotics are one of the most common antibiotics in the treatment of bacterial infections. However, the development of drug resistance against those medicines is becoming a serious concern. Aim This study aimed to develop an efficient, rapid, accurate, and sensitive detection method that is applicable for routine clinical use. Methods Escherichia coli was used as a model organism to develop a rapid, accurate, and reliable multiplex polymerase chain reaction (M-PCR) for the detection of four aminoglycoside modifying enzyme (AME) resistance genes Aac(6′)-Ib, Aac(3)-II, Ant(3″)-Ia, and Aph(3′)-Ia. M-PCR was used to detect the distribution of AME resistance genes in 237 clinical strains of E. coli. The results were verified by simplex polymerase chain reaction (S-PCR). Results Results of M-PCR and S-PCR showed that the detection rates of Aac(6′)-Ib, Aac(3)-II, Ant(3″)-Ia, and Aph(3′)-Ia were 32.7%, 59.2%, 23.5%, and 16.8%, respectively, in 237 clinical strains of E. coli. Compared with the traditional methods for detection and identification, the rapid and accurate M-PCR detection method was established to detect AME drug resistance genes. This technique can be used for the clinical detection as well as the surveillance and monitoring of the spread of those specific antibiotic resistance genes.

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Establishment of loop‑mediated isothermal amplification for rapid detection of Pseudomonas aeruginosa

Cited by 8 publications

References 20 publications

Detection of Mycobacterium tuberculosis in clinical sputum by a unique gene in MTB strains called Conserved protein TB18.5 (TB18.5)

Detection of Mycobacterium tuberculosis in clinical sputum by a unique gene in MTB strains called Conserved protein TB18.5 (TB18.5)

Rapid Visualization and Detection of Staphylococcus Aureus Based on Loop-Mediated Isothermal Amplification

A rapid and accurate method for the detection of four aminoglycoside modifying enzyme drug resistance gene in clinical strains of Escherichia coli by a multiplex polymerase chain reaction

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