Multiplex Isothermal Amplification Research Articles

Engineered G-Quadruplex-Embedded Self-Quenching Probes Regulate Single Probe-Based Multiplex Isothermal Amplification to Light Road Lamp Probes for Sensitized Determination of microRNAs

Design of oligonucleotide probe-based isothermal amplification with the ability to identify miRNA biomarkers is crucial for molecular diagnostics. In this work, we engineered a miRNA-21-responsive G-quadruplex-embedded self-quenching probe (GE-SQP) that can regulate single probe-based multiplex amplifications. The free GE-SQP is tightly locked in a quenching state with no active G-quadruplexes. Introduction of target miRNA to hybridize with GE-SQP would induce a multiplex isothermal amplification to significantly build a lot of one-bulb-contained road lamp probe (OC-RLP) and two-bulb-contained road lamp probe (TC-RLP) using G-quadruplex as the lamp bulb. When lightened by thioflavin T (ThT), beams of fluorescence were emitted to show the presented miRNA-21. Specially, the whole amplification is only a one probe-involved one-step reaction without any wasted species. The mix-to-detection and all-in amplification behavior allows the sensing system a maximally maintained operation simplicity and high assay performance. In such a way, the detection range of miRNA-21 is from 1 fM to 1 nM with a limit of detection of 0.86 fM. The practicability was demonstrated by determining miRNA-21 from serum samples with acceptable results. We expect that this method can open a new avenue for exploring advanced biosensors with improved analytical performances.

Development of a multiplex isothermal amplification molecular diagnosis method for on-site diagnosis of influenza.

Influenza, which is an acute respiratory disease caused by the influenza virus, represents a worldwide public health and economic problem owing to the significant morbidity and mortality caused by its seasonal epidemics and pandemics. Sensitive and convenient methodologies for the detection of influenza viruses are important for clinical care and infection control as well as epidemiological investigations. Here, we developed a multiplex reverse transcription loop-mediated isothermal amplification (RT-LAMP) with quencher/fluorescence oligonucleotides connected by a 5′ backward loop (LF or LB) primer for the detection of two subtypes of influenza viruses: Influenza A (A/H1 and A/H3) and influenza B. The detection limits of the multiplex RT-LAMP assay were 103 copies and 102 copies of RNA for influenza A and influenza B, respectively. The sensitivities of the multiplex influenza A/B/IC RT-LAMP assay were 94.62% and 97.50% for influenza A and influenza B clinical samples, respectively. The specificities of the multiplex influenza A/B/IC RT-LAMP assay were 100% for influenza A, influenza B, and healthy clinical samples. In addition, the multiplex influenza A/B/IC RT-LAMP assay had no cross-reactivity with other respiratory viruses.

A novel multiplex isothermal amplification method for rapid detection and identification of viruses.

A rapid multiplex isothermal amplification assay has been developed for detection and identification of multiple blood-borne viruses that infect millions of people world-wide. These infections may lead to chronic diseases or death if not diagnosed and treated in a timely manner. Sets of virus-specific oligonucleotides and oligofluorophores were designed and used in a reverse-transcription loop-mediated multiplexed isothermal amplification reaction for detection and gel electrophoretic identification of human Immunodeficiency virus (HIV), hepatitis-B virus (HBV), hepatitis-C virus (HCV), hepatitis-E virus (HEV), dengue virus (DENV), and West Nile (WNV) virus infection in blood plasma. Amplification was catalyzed with two thermostable enzymes for 30–60 minutes under isothermal condition, utilizing a simple digital heat source. Electrophoretic analysis of amplified products demonstrated simultaneous detection of 6 viruses that were distinctly identified by unique ladder-like banding patterns. Naked-eye fluorescent visualization of amplicons revealed intensely fluorescing products that indicated positive detection. The test demonstrated a 97% sensitivity and a 100% specificity, with no cross-reaction with other viruses observed. This portable detection tool may have clinical and field utility in the developing and developed world settings. This may enable rapid diagnosis and identification of viruses for targeted therapeutic intervention and prevention of disease transmission.

Simultaneous Detection of RNA and DNA Targets Based on Multiplex Isothermal Amplification

The detection of pathogenic microorganisms present in food, feed, plant, and other samples is important for providing safe food as well as for preventing the spread of microbes. The genome of pathogens is made of DNA or RNA, therefore a multiplex diagnostics tool would ideally be able to amplify and detect both RNA and DNA targets in parallel. With this goal we have developed an isothermal nucleic acid sequence based amplification [NASBA] implemented microarray analysis (NAIMA) procedure, suitable for the simultaneous multiplex amplification of RNA and DNA targets, coupled with the detection on ArrayTubes. The method is demonstrated to be very sensitive and specific for the detection of two economically important quarantine plant pathogens of potato, the potato spindle tuber viroid (RNA target) and Ralstonia solanacearum (DNA target). Because of its isothermal amplification and simple detection equipment, the method is also applicable for on-site analyses. NAIMA can be used in any domain where there is the need to detect RNA and DNA targets simultaneously.

Multiplex isothermal solid-phase recombinase polymerase amplification for the specific and fast DNA-based detection of three bacterial pathogens.

We report on the development of an on-chip RPA (recombinase polymerase amplification) with simultaneous multiplex isothermal amplification and detection on a solid surface. The isothermal RPA was applied to amplify specific target sequences from the pathogens Neisseria gonorrhoeae, Salmonella enterica and methicillin-resistant Staphylococcus aureus (MRSA) using genomic DNA. Additionally, a positive plasmid control was established as an internal control. The four targets were amplified simultaneously in a quadruplex reaction. The amplicon is labeled during on-chip RPA by reverse oligonucleotide primers coupled to a fluorophore. Both amplification and spatially resolved signal generation take place on immobilized forward primers bount to expoxy-silanized glass surfaces in a pump-driven hybridization chamber. The combination of microarray technology and sensitive isothermal nucleic acid amplification at 38 °C allows for a multiparameter analysis on a rather small area. The on-chip RPA was characterized in terms of reaction time, sensitivity and inhibitory conditions. A successful enzymatic reaction is completed in <20 min and results in detection limits of 10 colony-forming units for methicillin-resistant Staphylococcus aureus and Salmonella enterica and 100 colony-forming units for Neisseria gonorrhoeae. The results show this method to be useful with respect to point-of-care testing and to enable simplified and miniaturized nucleic acid-based diagnostics. FigureThe combination of multiplex isothermal nucleic acid amplification with RPA and spatially-resolved signal generation on specific immobilized oligonucleotides Electronic supplementary materialThe online version of this article (doi:10.1007/s00604-014-1198-5) contains supplementary material, which is available to authorized users.