Europium Nanoparticle-Based Lateral Flow Strip Biosensors Combined with Recombinase Polymerase Amplification for Simultaneous Detection of Five Zoonotic Foodborne Pathogens.

The five recognized zoonotic foodborne pathogens, namely, Listeria monocytogenes, Staphylococcus aureus, Streptococcus suis, Salmonella enterica and Escherichia coli O157:H7, pose a major threat to global health and social-economic development. These pathogenic bacteria can cause human and animal diseases through foodborne transmission and environmental contamination. Rapid and sensitive detection for pathogens is particularly important for the effective prevention of zoonotic infections. In this study, rapid and visual europium nanoparticle (EuNP)-based lateral flow strip biosensors (LFSBs) combined with recombinase polymerase amplification (RPA) were developed for the simultaneous quantitative detection of five foodborne pathogenic bacteria. Multiple T lines were designed in a single test strip for increasing the detection throughput. After optimizing the key parameters, the single-tube amplified reaction was completed within 15 min at 37 °C. The fluorescent strip reader recorded the intensity signals from the lateral flow strip and converted the data into a T/C value for quantification measurement. The sensitivity of the quintuple RPA-EuNP-LFSBs reached a level of 101 CFU/mL. It also exhibited good specificity and there was no cross-reaction with 20 non-target pathogens. In artificial contamination experiments, the recovery rate of the quintuple RPA-EuNP-LFSBs was 90.6-101.6%, and the results were consistent with those of the culture method. In summary, the ultrasensitive bacterial LFSBs described in this study have the potential for widespread application in resource-poor areas. The study also provides insights in respect to multiple detection in the field.

Colorimetric Detection of 23 Human Papillomavirus Genotypes by Loop-Mediated Isothermal Amplification.

BACKGROUND: Human papillomavirus (HPV) infection is linked to cervical cancer. With the technological development of molecular biology and epidemiology, detection and treatment of HPV has become an important mean to prevent cervical cancer. METHODS: A simple, rapid, and sensitive colorimetric loop-mediated isothermal amplification (LAMP) method was established herein to detect 23 HPV genotypes. The sequences of the primers for the LAMP reaction were located in the L1 gene of the HPV genome. As it is a fluorescent dye, calcein was added before the reaction. The reaction was run under isothermal conditions at 65°C for 40 minutes. A positive reaction was indicated by a color change from yellow to fluorescent green. The fluorescence curve diagram represents the monitoring of real time quantitative instrument. 450 cervical swab samples from patients with single infections of 23 different HPV genotypes were examined to evaluate the specificity. RESULTS: The results revealed no cross-reaction with other HPV genotypes. A serial dilution of a cloned plasmid containing 23 HPV L1 gene sequences was employed to evaluate the sensitivity. Different HPV subtypes have different detection capability. The sensitivity of different HPV subtypes tested by LAMP assay was in the range from 1.0 x10 to 4.0 x 103 copies per reaction. The LAMP assay and the RDB (reverse dot blot) were compared for detecting and genotyping HPV among the 450 clinical samples. There were 385 (85.6%) and 375 (83.3%) HPV positive specimens detected by LAMP and RDB, respectively, as well as 306 (68.0%) and 296 (65.8%) for HR-HPV positive specimens. The agreement between the LAMP and RDB assays was 93.3% (κ = 0.75) for HPV positivity and 94.7% (κ = 0.88) for HR-HPV positivity. CONCLUSIONS: It was concluded that this colorimetric LAMP assay had potential application for the rapid screening of the HPV infection in resource-limited hospitals or rural clinics.

Isothermal Method of a Recombinase Polymerase Amplification Assay for the Detection of Most Common High-Risk Human Papillomavirus Type 16 and Type 18 DNA.

BACKGROUND: Cervical cancer is a common gynecologic malignant tumor and has a great impact on women's health. Human papillomavirus (HPV) is implicated in cervical cancer and precancerous lesions and the two are possibly two stages of disease progression. With the technological development of molecular biology and epidemiology, detection and treatment of HPV has become an important means to prevent cervical cancer. METHODS: Here we present a novel, rapid, sensitive and specific isothermal method of recombinase polymerase amplification (RPA), which is established to detect the two most common high-risk human papillomavirus type 16 and type 18 DNA. In this study, we evaluate the efficacy of the RPA assay, incubating clinical specimens of HPV16 and HPV18 using plasmids standard. It operates at constant low temperature without the thermal instrumentation for incubation. The products can be detected via agarose gel electrophoresis assay, reverse dot blot assay, and quantitative real-time assay with SYBR Green I. We assess the diagnostic performance of the RPA assay for detecting of HPV16 and HPV18 in 335 clinical samples from patients suspected of cervical cancer. RESULTS: The results revealed no cross-reaction with other HPV genotypes and the RPA assay achieve a sensitivity of 100 copies. Compared with TaqMan qPCR, the RPA technique achieves exponential amplification with no need for pretreatment of sample DNA at 37°C for 20 minutes, which reveals more satisfactory performance. The agreement between the RPA and qPCR assays was 97.6% (κ = 0.89) for HPV16 positivity and 98.5% (κ = 0.81) for HPV18 positivity, indicating very good correlation between both tests. CONCLUSIONS: Importantly, the RPA assay was demonstrated to be a useful and powerful method for detection of HPV virus, which therefore may serve as a valuable tool for rapid diagnosis of HPV infection in both commercial and clinical applications.