Real-time Fluorescence Loop-Mediated Isothermal Amplification Technology For Rapid Detection Of Proteus

Proteus is a food-borne pathogen that can easily contaminate meat, animal offal, eggs, and other animal food, as well as salad, leftovers, and soy products; Proteus can cause acute gastroenteritis or histamine poisoning and thus results in decreased body resistance, which eventually leads to meningitis, peritonitis, septicemia, urinary tract infections, respiratory infections, rheumatoid arthritis, and other diseases. The traditional method for routine detection of Proteus is complex, time-consuming, and cannot meet the rapid and sensitive detection requirements of pathogens in food. Therefore, developing a rapid detection method for Proteus is very important.we investigated real-time fluorescence loop-mediated isothermal amplification(Real Amp) technology using a simple and portable fluorescence reader(ESE-Quant Tube Scanner) that capable of performing both the amplification and detection of LAMP by connecting to a computer in one platform. A type of fluorescent dye(SYBR Green Ⅰ) was added in the LAMP reaction system. Fluorescence intensity is significantly enhanced after SYBR GreenⅠ binds to the double-stranded DNA; thus, as the reaction proceeds, the amount of DNA increases, and the fluorescence intensity of the reaction system also changes. The higher the peak, the higher the efficiency of LAMP amplification reaction. The Real Amp method can be used for real-time monitoring and intuitive judgment of the test results.Four primers were constructed using an online primer designer(https:// primerexplorer. jp/lamp3.0.0/index. html) within the six areas of the target gene; the primers were based on the atp D gene sequences(P. vulgaris: AY134488; P. mirabilis: AX109601) of Proteus published in Gen Bank and were as follows: forward outer primer(F3), backward outer primer(B3), forward inner primer(FIP), and backward inner primer(BIP). The portable fluorescence reader(ESE-Quant Tube Scanner) was used for isothermal(62 ℃) amplification of DNA template. Real-time monitoring and judgment of test results were achieved by connecting the reader to a computer. The sensitivity and specificity of this system were determined and compared with those of the conventional LAMP. Additionally, the detection limit of artificially contaminated pork was determined using Real Amp. Moreover, the obtained Real Amp products were subjected to HpaⅠ restriction analysis to verify their specificities.The reaction system and response conditions of Real Amp were determined with ESE-Quant Tube Scanner after repeated optimization. Reaction system consisted of 5 μL of high-purity d NTPs(2.5 mmol/L), 3 μL of 10 × Thermo Pol TM reaction buffer, 0.5 μL of Mg SO4(50 mmol/L), 1 μL of F3 and B3(10 μmol/L), 2.5 μL of FIP and BIP(10 μmol/L), 0 μL of betain(5 mol/L), 1.5 μL of Bst DNA polymerase large fragment(8000 U/m L), 1.5 μL of SYBR Green I(1: 500 dilution), and 3 μL of template DNA. The total volume was 25 μL, which was complemented with sterile distilled water. Reaction condition included amplification at 62 ℃ for 60 min. The reaction can be terminated in accordance with the actual reaction conditions. Real Amp method was more simple and rapid than the conventional LAMP, The total time of judgment result of the former was generally less than 25 min, however, the latter need to spend about 2 h. A total of 19 strains were subjected to specificity tests, 6 Proteus strains indicated positive results, whereas the other 13 non-Proteus strains indicated negative results. The sensitivity of Real Amp in detecting pure cultures of Proteus was 8.1 CFU/m L compared with that of the conventional LAMP at 81 CFU/m L. Moreover, the detection limit of artificially contaminated pork was 81 CFU/m L. The obtained Real Amp products were subjected to Hpa I restriction analysis to verify their specificities, the measured restriction fragment size is consistent with the theoretical value.Real Amp method developed in this study can be real-time monitoring and visually determine test results, which is superior in terms of testing time, convenience, specificity and sensitivity; thus, Real Amp has significant potential for rapid detection of Proteus. There are conducive to the application of inspection and quarantine agencies grassroots...