Colorimetric Strategy Coupled With Isothermal Nucleic Acid Amplification Technique For The Detection Of Nosema Bombycis

As an important part of China’s agriculture,the products of the silkworm industry are not only used as raw materials for the silk industry,but also are used for food and medicine.Nosema bombycis is a kind of microscopic single-celled fungus parasitic on the intestinal wall of silkworm.Its infection can lead to weight loss,loss of appetite,slow action,and even death of silkworm,causing huge economic losses to sericulture.Therefore,it has been listed as the legal quarantine target in sericulture production by various sericulture countries and regions.In this study,a quantitative method for nucleic acid amplification products analysis was obtained by revealing the visual sensing mechanism of hydroxynaphthol blue,and a portable colorimetric instrument for in-situ detection of amplification results in the reaction tube was developed.Further,a simple and rapid method for the detection of small subunit ribosomal RNA（SSU r RNA）gene of the Nosema bombycis was developed by using the loop-mediated isothermal amplification（LAMP）technique.The main results of the study are as follows:（1）The visual sensing mechanism of hydroxynaphthol blue was revealed,and a simple colorimetric method for the quantitative analysis of nucleic acid amplification products was obtained.The effects of Mg²⁺ions,deoxynucleoside triphosphates（d NTPs）and byproduct Mg₂P₂O₇ on hydroxynaphthol blue were analyzed by UV absorption spectrum.The results showed that the interaction between hydroxynaphthol blue and Mg²⁺ions leads to the absorption of red light at 648 nm,and the absorption increased with the consumption of Mg²⁺ions.The color change of hydroxynaphthol blue was proportional to the absorbance at648 nm with the equation of y=17.54x+0.0042（R²=0.9979）,which was used to quantify the color change of hydroxynaphthol blue.Secondly,the chelation between d NTPs and Mg²⁺ions enhanced the linear correlation between hydroxynaphthol blue and Mg²⁺ions,which was beneficial to the quantitative detection of Mg²⁺ions consumption.In addition,Mg₂P₂O₇presented in the solution did not interfere with the detection,and it improved the sensitivity of the detection.Thus,a simple colorimetric method for the analysis of amplified products was obtained with the formula of y=0.1895x+0.0631（R²=0.9809）.（2）In-situ analysis of colorimetric signal generated by hydroxynaphthol blue.To avoid the high risk of aerosol contamination,we firstly simplified the complicated optical system of colorimetric instrument by using an light-emitting diode（LED）and an light-dependent resistor（LDR）.After that,the effects of cuvette shape on the detection were investigated,and a portable instrument using PCR tubes as the cuvette was developed for in-situ analysis without uncapping operation.The detection of Mg²⁺inos consumption showed a linear responds with the equation of y=0.1873x+0.0005（R²=0.9932）.It made the analysis more simple and rapid.Moreover,it could reduce the high risk of aerosol contamination and false-positive results that may be caused by the uncapping operation.（3）The rapid method for the Nosema bombycis detection.Firstly,LAMP primers were designed by using the SSU r RNA gene of Nosema bombycis as the target.After the amplification of 30 min at 66°C,a linear range between 10⁰～10⁴ copiesμL^-1 was obtained with the equation of y=0.0988 lg c+0.1517（R²=0.9967）,and the detection limit was 10⁰copyμL^-1 The recoveries of spiked silkworm egg samples were between 86.1%and 93.0%with all RSDs lower than 2.0%,suggesting the feasibility of the developed method for the quantitative and rapid detection of Nosema bombycis.In this study,we developed a simple and economical method for the rapid analysis of amplified products,and further achieved rapid and quantitative detection of Nosema bombycis,providing a useful and general approach for the rapid detection of pathogenic microorganisms to ensure food safety.