Study On A Novel Method For Constant Temperature Amplification Of Nucleic Acid And Its Application In Field Detection

Since Mullis invented Polymerase Chain Reaction(PCR) in the 80’s of last century, molecular biology has entered a new era of rapid development. Various vitro nucleic acid amplification technologies also came into being, such as fluorescence quantitative PCR, nested PCR, reverse transcription PCR and so on. However, just like PCR, these technologies belong to temperature-programmed reactions. Thus, a strict temperature controlled and sophisticated equipment is needed.Isothermal Nucleic Acid Testing(INAT) is a kind of nucleic acid amplification technologies which can be performed under an isothermal temperature. Because of the single temperature requirement, the INAT reactions can be carried out in a constant temperature instrument. A water bath, metal bath or even a cup with fine insulation effect is sufficient to complete the reaction. INAT technologies completely abandon the temperature-programmed devices required by PCR and greatly simplify the operation steps. The professional level requirement is relatively low for INAT. INAT meets the requirement of point-of-care testing(POCT), such as in scene of the field army, community-based medical, field epidemiological investigation, and the sickbed-bedside diagnosis.INAT technology mainly includes Strand displacement amplification(SDA), Rolling circle amplification(RCA), Loop-mediated isothermal amplification(LAMP), Recombinase polymerase amplification(RPA) and so on. The LAMP method with high amplification efficiency requires only one kind of enzyme, is currently the mostly applied technique within INAT. However, LAMP also has some shortcomings of complex primer design, high false-positive rate and high reagent price. Moreover, because of Japan’s protection of its intellectual property, there’re limitations in LAMP application in our country.In this study, we invented a new nucleic acid amplification technology, termed Polymerase Spiral Reaction(PSR). PSR utilizes the design of hybrid primers and a Bst DNA polymerase with strand displacement activity. Through the cycle of primer annealing, extension, circular, single stranded rotation and extension, the purpose of nucleic acid amplification in isothermal conditions is achieved. PSR has the advantages of simple primer design like PCR method and the LAMP method which needs only one kind of enzyme and has high amplification efficiency. It is the first time to realize nucleic acid amplification using one pair of primers and one kind of enzyme in isothermal environment by PSR. Moreover, in order to improve the reaction rate of PSR method, we introduced the concept of accelerating primer, and the Ct value of the reaction could be reduced to 20 min by adding the accelerating primer, and the reaction could be completed within 30 min. In addition, in this study, the aerosol pollution problem was solved through the use of liquid and solid sealant, the reaction system and primer design parameters were also optimized to eventually establish the integrated nucleic acid isothermal amplification method.Commonly used result interpretation methods for nucleic acid testing include agarose gel electrophoresis, fluorescence imaging method, layer chromatography test strip and real-time turbidity method. However, all of these methods have a common characteristic that the need of professional instrument, and the high operator’s professional level. Based on the changes of metal ion concentration and pH value of reaction system in PSR assay, three color indicator, calcein, hydroxy naphthol blue(HNB) and pH value indicator(cresol red and phenol red), were included to evaluate colorimetric method. A simple color change can be interpreted to determine the reaction negative or positive. The color for negative result is pale orange for calcein, purple for HNB and red for pH value indicator respectively. The color for positive result is green for calcein, sky blue for HNB and yellow for pH value indicator respectively. The colorimetric method is simple and economic which can be identified by naked eye, fully meets the requirements of POCT detection.DNA extraction from biological samples is the first step for nucleic acid detection. It is also indispensable for POCT situations such as field and clinical detection. Generally speaking, the nucleic acid extraction process relies on different kind of kits using guanidine salt lysis method, silicon membrane or paramagnetic particle method, and nuclear acid automatic extraction instrument. However, the process of nucleic acid extraction using kits is time-consuming and complicated to operate, which usually needs more than 2 hours. The nuclear acid automatic extraction instrument can realize high-throughput automation and simple-operation. But it is relatively bulky and high costly for maintenance. Relying on nucleic acid exposure theory, this study analyzed the effect of nonionic surfactants Chelex-100 and TritonX-100 on DNA extraction from different pathogenic microorganisms, including gram positive bacteria, gram negative bacteria, fungi, and viruses. The Chelex lysis buffer has the characteristics of simple operation, equivalent lysis effect as commercial kits and does not affect the subsequent reaction, especially suitable for POCT environments.The number of emerging infectious disease outbreaks has been increasing in recent years, does great harm to people’s lives and safety. After the establishment of PSR reaction and corresponding biological sample processing and fast result interpretation method, we built a technological platform for rapid detection based on PSR reaction. The platform provides technical support for daily disease prevention tasks, laboratory detection for routine pathogens, batch application from national food and drug administration, and countermeasures against emerging infectious disease outbreaks.PSR detection methods were also established for six kinds of pathogenic microorganisms, Klebsiella pneumoniae, Pseudomonas aeruginosa, Candida albicans, Enterovirus 71 and Coxsackievirus A16, Vibrio cholera in this study. The experimental evaluation confirmed that the PSR assay has high specificity and sensitivity. The PSR detection methods were further used for clinical detection and a series of fine results were achieved.Eventually, we conducted technical transformation for PSR and its related technologies. Through exchanges and cooperation with domestic counterparts, we adopted a vitrification technology to treat the bioactive substances in the reaction buffer, such as enzyme and primers, to solve the problems of easy deactivation in non-cryopreservation environment. We developed corresponding sample processing pipe for Chelex lysis buffer, which has four features of pathogen inactivation, nucleic acid releasing, impurity filtering, and precise sampling. In addition, this study designed a detection instrument with both the lysis zone and amplification zone. Through extrusion the processing pipe a drop of lysis buffer was added to the reaction tube cover. After a short moment, the drop was swung to the bottom of reaction tube and subjected to the detecting instrument. After prompting sound out, we can easily interpret the result negative or positive through the color change. The detection instrument can handle complex samples of blood, stool, urine, etc.In conclusion, this study invented a novel isothermal nucleic acid amplification method, termed polymerase spiral reaction, and introduced new methods for sample DNA extraction and result interpretation for amplification. We also established a technological platform for rapid detection based on PSR method and conducted technical transformation. The PSR assay is characterized by speediness, efficiency, high specificity and high sensitivity. We believe that PSR technology will provide strong technical supports to deal with emerging infectious diseases outbreaks and laboratory routine pathogen detection. The PSR method will be gradually introduced to the basic level places, to the families, and finally validate nucleic acid testing everywhere.