Study On The Determination Of Cytochrome Cd1-containing Nitrite Reductase Encoding Gene NirS By Loop-mediated Isothermal Amplification Assay

Denitrification is a dissimilatory process in which oxidized nitrogen is reduced to nitrous oxide or nitrogen.As a critical part of the global nitrogen cycle,it is responsible for the return of fixed nitrogen back to the atmosphere.There are 4 steps in the process,namely nitrate reduction,nitrite reduction,nitric oxide reduction and nitrous oxide reduction,catalyzing by nitrate reductase,nitrite reductase,nitric oxide reductase and nitrous oxide reductase,respectively.Among these steps,the nitrite reduction is the rate-controlling one.Therefore,the determination of cytochrome cd1-containing nitrite reductase encoding gene nir S is a useful approach to understand the distribution and potential activity of denitrifying bacteria and denitrifier communities,as well as the translating law of nitrogen element in ecological environment.The data also are essential for assessing the state of ecological health in the fields of scientific research and other relevant activities.Up to now,quite a few methods,such as polymerase chain reaction(PCR),denaturing gradient gel electrophoresis and gene chips,have been constructed for the determination of target genes.Most of them are highly sensitive and accurate.However,their applications are limited in rapid and point-of-care measurements due to the requirements of specialized laboratories,trained operators and expensive instruments.In addition,the stress of cost is hard to be addressed.Loop-mediated isothermal amplification(LAMP),which amplifies DNA under isothermal conditions,requires a set of four or six specially designed primers that recognize six or eight distinct regions of target genes.The elimination of template cycle makes it a potential to revolutionize molecular biology by reducing the need for highly sophisticated equipment,and by having low running costs and short turnaround times compared with many amplification methods.It exhibits higher sensitivity,amplification efficiency and specificity than that of PCR.Moreover,the reaction of LAMP is also more tolerant to well-known PCR inhibitors such as blood,serum and food ingredients.These attractive properties have motivated researchers to explore the use of LAMP for genetic analysis in diverse fields,particularly,for point-of-care uses.There are two key studies in this thesis.One is the development of LAMP assay for determining nirS gene of Pseudmonas aeruginosa PAO1.The other is the demonstration of why the target gene can be amplified with LAMP using viable bacterial cells as template directly.A new molecular biology method was developed for determining rapidly nirS gene of Pseudmonas aeruginosa PAO1,by employing LAMP assay.The LAMP assay relied on a set of four primers that were designed to recognize six target sequence sites,resulting in high target specificity.The specificity of the assay was confirmed by the lack of amplification when using DNA from 15 other standard and isolated bacterial species,including Escherichia coli,Staphylococcus aureus,etc.Using gel electrophoresis or fluorescent dye GeneFinder to detect the LAMP products,the limit of detection(LOD)for the assay under optimized conditions was 1.87 pg/reaction of genomic DNA,which was an order of magnitude lower than that required by conventional PCR assays.Moreover,a cell-template based LAMP assay was also developed for detecting nirS gene that directly used viable bacterial cells as template rather than genomic DNA.The LOD for the assay under optimized conditions was3.36×10~2 CFU/mL.By contrast,PCR assay could not be used to achieve the determination by directly using viable cells as template.For the LAMP assay,only 1 h at 63?was needed from the addition of genomic DNA or bacterial cells to the reaction mix to the verification of amplification success,and bulky and sophisticated equipment were not needed.Further,the nirS gene of Pseudmonas aeruginosa PAO1in spiked seawater samples could be determined with both the DNA-template based LAMP assay and the cell-template based LAMP assay,thereby demonstrating the practicality of in-field use.In summary,the LAMP assays described here represent a rapid,user-friendly,and cost-effective alternative to conventional PCR.When the LAMP assay is used for detecting target genes,DNA extraction is unnecessary in many cases.Simple pretreatment and even viable bacterial cells without any pretreatment can be used as template.Using stx1 gene from E.coli as model,we verified that viable cells,dead cells and extracellular DNA could function as template in the LAMP assay.In the incubation at 63°C,viable bacteria in the LAMP reaction mixture lysed completely within 2 min,providing DNA template for nucleic acid amplification.The stx1 gene in diluted culture medium,spiked tap water,spiked seawater and real seawater all could be detected,with or without the step of DNA extraction.We found that the complex substances in real sample(e.g.natural seawater)exhibited considerable inhibitory effect on the sensitivity of the LAMP assay.These outcomes are meaningful for building a point-of-care strategy by employing the LAMP assay for environmental monitoring,bio-resource surveys,food safety,etc.in particular those based on environmental DNA.