RETRACTED ARTICLE: Polymerase Spiral Reaction (PSR): A novel isothermal nucleic acid amplification method

Abstract

In this study, we report a novel isothermal nucleic acid amplification method only requires one pair of primers and one enzyme, termed Polymerase Spiral Reaction (PSR) with high specificity, efficiency, and rapidity under isothermal condition. The recombinant plasmid of blaNDM-1 was imported to Escherichia coli BL21, and selected as the microbial target. PSR method employs a Bst DNA polymerase and a pair of primers designed targeting the blaNDM-1 gene sequence. The forward and reverse Tab primer sequences are reverse to each other at their 5’ end (Nr and N), whereas their 3’ end sequences are complementary to their respective target nucleic acid sequences. The PSR method was performed at a constant temperature 61 °C–65 °C, yielding a complicated spiral structure. PSR assay was monitored continuously in a real-time turbidimeter instrument or visually detected with the aid of a fluorescent dye (SYBR Greenı), and could be finished within 1 h with a high accumulation of 109 copies of the target and a fine sensitivity of 6 CFU per reaction. Clinical evaluation was also conducted using PSR, showing high specificity of this method. The PSR technique provides a convenient and cost-effective alternative for clinical screening, on-site diagnosis and primary quarantine purposes.

Establishment of an accurate and fast detection method using molecular beacons in loop-mediated isothermal amplification assay

“…Attempts to pair fluorescent probes with isothermal amplification methods have been previously successful, such as the use of fluorescence energy transfer probes with strand displacement amplification (SDA), molecular beacons with nucleic acid sequence-based amplification (NASBA), and padlock probes with rolling circle amplification (RCA)171819. Based on these techniques and the outcome of the current experiments, MB-LAMP could also be extended to most isothermal amplification methods, such as polymerase spiral reaction (PSR)20, cross-priming amplification(CPA)21, transcript-based amplification system (TAS)22, and single primer isothermal amplification (SPIA)23.…”

Visual detection of porcine epidemic diarrhea virus using a novel reverse transcription polymerase spiral reaction method

“…The RT-PSR products with repeat target sequences displayed multiple banding patterns were produced due to different spiral amplification stages by the aid of simultaneous Bst DNA polymerase extension at 3′ end and strand displacement at 5′ end. [14].…”

“…Detection methods based on isothermal amplification of nucleic acids, which can rapidly synthesize large amounts of DNA without any specific requirements for precision instruments, have been widely used. The polymerase spiral reaction (PSR) [14] is a novel nucleic acid isothermal amplification method that has the advantages of simplicity, rapidity, accuracy, and low cost when compared to conventional PCR. In addition, less primer is required, and primer design is simpler than for loop-mediated isothermal amplification (LAMP).…”

Isothermal nucleic acid amplification techniques for detection and identification of pathogenic fungi: A review

“…Since 90s, many isothermal nucleic acid amplification techniques (INAT) arose in order to make the molecular methods affordable to clinical laboratories in developing countries, since these techniques do not require specific apparatus, as thermocycler, electrophoresis devices and, sometimes, UV light source, which are expensive equipments that usually demand maintenance costs as well 7 . The main INATs developed and applied for the diagnosis of infectious diseases are nucleic acid sequence‐based amplification (NASBA), 8 rolling circle amplification (RCA), 9 loop‐mediated isothermal amplification (LAMP), 10 helicase‐dependent amplification (HDA), 11 cross‐priming amplification (CPA) 12 and polymerase spiral reaction (PSR) 13 . To allow an isothermally nucleic acid amplification, new strategies have been used by performing all steps of DNA denaturation, primer hybridisation and polymerase amplification under the same temperature and, therefore, independent of a thermocycler.…”