Establishment And Optimization Of Loop-mediated Isothermal Amplification(LAMP) For Rapid Identification Of Morinda Officinalis

Objective:Morinda officinalis how（MO）,a traditional Chinese medicine,has been widely used as a therapeutic medicine and popular tonic in China or around the world.The appearances of the easily confused medicinal herbs are similar to MO,so it is hard and inconvenient to distinguish them by conventional methods.In this study,the loop-mediated isothermal amplification（LAMP）method was established to identify MO by designed a set of LAMP primers,then determined the reaction system and optimizing the conditions,so as to provide a new basis for the molecular detection of Chinese medicinal materials.Methods:The MO,Morinda umbellata L.,Schisandra propinqua subsp.Sinensis,Damnacanthus indicus C.F.Gaertn,Morinda parvifolia,Morinda citrifolia Linn,Akebia trifoliata subsp.Australis,Kadsura longipedunculata Finet et Gagnep were collected for DNA extraction.The DNA was quantitatively and screened by spectrophotometer,then adjusted to the concentration of 10 ng/μL by TE buffer solution and stored at-20℃for further usage.After multiple sequence alignment,the internal transcribed spacer 2（ITS2）sequence of MO was used as the template to design and select the optimal primers for establishing LAMP detection method,then test the repeatability,specificity and sensitivity of this method.The DNA extracted from MO and other herbs were detected with the same experimental condition,the specificity is excellent if only MO could amplify successfully;The gene template of MO was diluted in serial gradients and compared with the conventional PCR method to determine and evaluate the sensitivity.In order to further improve the detection effect,Response Surface Methodology was used to optimize the temperature and reaction system which influence the highest fluorescence intensity most.Firstly,according to the experimental result of Plackett-Budrman experiment,three factors affecting the fluorescence intensity most were selected,and the horizontal step path is designed based on its change direction as the climbing direction.Next,the response surface analysis experiment with three factors and three levels is designed based on the central combination principle of Box-Behnken.Finally,the statistical significance of the regression model was analyzed and a reaction condition which can reach the highest fluorescence intensity was obtained with verification.Results:Combined with DNA barcodes technology,a set of LAMP primers which could identify MO specificity were designed based on ITS2 sequence.LAMP reaction can be completed in 40～60 minutes at 63℃,and the detection results can be achieved in 24 hours,one week,one month or six months.The three LAMP detection methods,including visual inspection method based on calcein color determination,agarose gel electrophoresis and real-time fluorescence quantitative method,have the advantages of simple,objective and convenient to quantify.The results of specific test showed that LAMP technology could amplify MO DNA as expected and distinguish it from other plant sequences with objective and obvious differences include color variance,electrophoretic stripes and amplification curves.The results of sensitivity test showed that the detection limit of this method was about 100 fg/μL in a concentration range of 10fg/μL～100pg/μL,which was more sensitive than PCR method.Through the significant screening of KCl,（NH₄）₂SO₄,Tris-HCl,d NTPs,Mg SO₄,FIP/BIP,F3/B3,Tween20,betaine,polymerase and temperature,the three factors that have the highest influence on fluorescence intensity are temperature,content of primers F3/B3 and Mg SO₄.After optimization by response surface methodology,it can be concluded that the fluorescence intensity of this method will reach the highest at the optimal condition of 63℃,5.02 pmol F3/B3 and 8.11 m M Mg SO₄.The verification results are consistent with the prediction results.Conclusion:Identification of MO by LAMP technology has the advantages of high accuracy,strong specificity and objective evaluation.This method has low equipment requirement,simple operation and fast speed,which expands the detection method of traditional Chinese medicine and has the potential of further commercial application.