The Direct Semi-Quantitative Detection of 18 Pathogens and Simultaneous Screening for Nine Resistance Genes in Clinical Urine Samples by a High-Throughput Multiplex Genetic Detection System

Abstract

BackgroundUrinary tract infections (UTIs) are one the most common infections. The rapid and accurate identification of uropathogens, and the determination of antimicrobial susceptibility, are essential aspects of the management of UTIs. However, existing detection methods are associated with certain limitations. In this study, a new urinary tract infection high-throughput multiplex genetic detection system (UTI-HMGS) was developed for the semi-quantitative detection of 18 pathogens and the simultaneously screening of nine resistance genes directly from the clinical urine sample within 4 hours.MethodsWe designed and optimized a multiplex polymerase chain reaction (PCR) involving fluorescent dye-labeled specific primers to detect 18 pathogens and nine resistance genes. The specificity of the UTI-HMGS was tested using standard strains or plasmids for each gene target. The sensitivity of the UTI-HMGS assay was tested by the detection of serial tenfold dilutions of plasmids or simulated positive urine samples. We also collected clinical urine samples and used these to perform urine culture and antimicrobial susceptibility testing (AST). Finally, all urine samples were detected by UTI-HMGS and the results were compared with both urine culture and Sanger sequencing.ResultsUTI-HMGS showed high levels of sensitivity and specificity for the detection of uropathogens when compared with culture and sequencing. In addition, ten species of bacteria and three species of fungi were detected semi-quantitatively to allow accurate discrimination of significant bacteriuria and candiduria. The sensitivity of the UTI-HMGS for the all the target genes could reach 50 copies per reaction. In total, 531 urine samples were collected and analyzed by UTI-HMGS, which exhibited high levels of sensitivity and specificity for the detection of uropathogens and resistance genes when compared with Sanger sequencing. The results from UTI-HMGS showed that the detection rates of 15 pathogens were significantly higher (P<0.05) than that of the culture method. In addition, there were 41(7.72%, 41/531) urine samples were positive for difficult-to-culture pathogens, which were missed detected by routine culture method.ConclusionsUTI-HMGS proved to be an efficient method for the direct semi-quantitative detection of 18 uropathogens and the simultaneously screening of nine antibiotic resistance genes in urine samples. The UTI-HMGS could represent an alternative method for the clinical detection and monitoring of antibiotic resistance.

MC-PRPA-HLFIA Cascade Detection System for Point-of-Care Testing Pan-Drug-Resistant Genes in Urinary Tract Infection Samples

“…A cascade detection system based on RPA was constructed for this study, which offers greater detection sensitivity than PCR-based tests. For the detection of pan-drug-resistant genes in UTI samples, the multiplex PCR-based assay had an LOD of 10 3 CFU/mL, which was inferior to the cascade assay [ 18 ]. The latest nanopore sequencing technology could also be used for the detection of these genes in UTI samples, with an accuracy comparable to that of commonly used clinical methods; however, this assay still requires pre-culture of UTI samples, which is time-consuming [ 19 ].…”

A rapid and high-throughput multiplex genetic detection assay for detection, semi-quantification and virulence genotyping of Helicobacter pylori in non-invasive oral samples

“…The gene sequences of H. pylori strain-specific identification gene 16S rRNA, single-copy semi-quantitative gene ureC, 10 virulence genes (vacA s1, vacA s2, vacA m2, vacA m1, cagA, oipA, luxS, iceA2, iceA1 and dupA) and one human internal reference gene b-globin were downloaded from the National Center of Biotechnology Information (NCBI). Vector NTI (Invitrogen, Carlsbad, USA) was used for multiple sequence alignment, and highly conserved specific sequences were selected to design primers (Sun et al, 2021). Next, primers were designed to amplify each of the highly conserved regions using DNASTAR (DNASTAR Inc., Madison, WI, USA) and Primer Premier 6.0 (Premier Biosoft International, Palo Alto, CA, USA) (Sun et al, 2021).…”

The Essential Role of PCR and PCR Panel Size in Comparison with Urine Culture in Identification of Polymicrobial and Fastidious Organisms in Patients with Complicated Urinary Tract Infections

“…Several pioneer studies have suggested that recently developed syndromic polymerase chain reaction (PCR) molecular assays have decreased turn-around time (TAT) and are more effective in identifying PMOs and FOs when compared to conventional UC [ 23 , 24 , 25 , 26 , 27 ]. These studies were limited as the patient population was not confined to those clinically identified with cUTIs.…”