Study And Evaluation Of A Novel Visualized Loop-mediated Isothermal Amplification (LAMP) For Plasmodium Detection

Malaria, which is caused by protozoan parasites of the genus Plasmodium, is one ofthe major infectious diseases，threatens people’s health and life seriously. Malaria isone of the three major global public health concerns, and also poses a serious threat tohealth and economic development in China Two large-scale malaria outbreaks in thecentral region of China during the early1960s and1970s, and this region hasexperienced vivax malaria resurgence since2000. Through unremitting efforts, Chinahas achieved great success in controlling malaria. Plasmodium falciparum has beenlargely eradiation in the central region since1990s, Rebound of vivax malaria andlocal outbreaks were controlled effectively in2009, and the incidence has dropped tohistorically low levels.According to the "Action Plan to Eliminate Malaria in China" which carried out in2010, there are big difference in goals, strategies and measures between two stages ofMalaria control and elimination. The goal of malaria control is to reduce the incidence,whereas no local case of malaria infection is for elimination. Therefore, accurate andtimely diagnosis of each possible infection source is the key point to prevent malariatransmission. The “Guide of Malaria Elimination” requires all of the suspect cases inmalaria eliminating stage should be diagnosed. Association with the decreasedmalaria incidence, parasitemia has declined and the percent of patients with lowerlevel of parasitemia is growing, which bring new challenges to malaria diagnosis. Traditional microscopic examination of blood smears was the most widely usedmalaria diagnostic method in laboratory, however, the shortcoming is its dependenceon the skills and experience of the microscopist and relatively low sensitivity,particularly at low parasite levels. Recently, Rapid Diagnosis Tests(RDTs) havebecome available, which are simple and convenient, but the sensitivity of thecurrently existing products to detect low parasitemia, especially P.v. infection, areusually limited. Molecular diagnosis such as nested PCR has the advantages of highsensitivity and specificity, yet it is involved with special reagents and equipmentswhich not only complex methodologies, high cost, but also the need for speciallytrained technicians, made it need to perform in well conditioned central lab currently.Loop-mediated isothermal amplification is novel method to detect DNA, which issimple to perform and have the great advantages of high sensitivity and specificity.LAMP reaction can be conducted under isothermal condition performed with astrand-displacement functional Bst DNA polymerase, and yield the results of109-1010fold amplification by only one step in a short time. With a by-product namedmagnesium pyrophosphate, the reaction solution contains white turbid which can beseen by naked eyes so that LAMP results can be identified according to the solutionhas turbid Whether or not. Adding dye calcein or SYBR Green I into the reaction mix.can improve the observation, however, the calcein lacks of sensitivity and the SYBRGreen I has a inhibition to the amplification made it needs to be added after reaction.Open the tube leads to high risks of products contamination due to the high efficiencyof LAMP.To solve the problem above, our lab already has developed a visualized LAMP todetect P.v. by adding a wax-dye capsule which had SYBR Green I inside. The dye can be released to solution after wax unmelted by heating, thus the products will bestained and identify by naked eyes，and then the wax re-solidify to become a barrier toavoid the contamination. However this visualized LAMP can detect P.v. only and itsdetection stability needs to be further improved. Considering besides the local P.v.infection in our country, there are also imported P.f., P.m. and P.o. cases of migrantworkers increased every year, most of which are in remote rural areas. A morestabilized, sensitive, special, inexpensive and simplified method which cansimultaneously detect4species of Plasmodium in high-throughput should bedeveloped for malaria diagnosis. In this research we modified and optimized thevisualized LAMP to detect Plasmodium. The benefit-cost between the modifiedvisualized LAMP with self-made reagents and the commercially available Loopamp DNA amplification kit was compared to estimate the usefulness in field. The modifiedvisualized LAMP was used to detect field blood filter DNA samples to evaluate it asan application in resource limited areas. The main content is listed as following.Part1Improvement of visualized LAMP in detecting PlasmodiumMethods: The conversation region of Plasmodium18S rRNA gene andmitochondrial gene were chosen as the target to design genus specific LAMP primersonline, together with those in papers are screened to yield a suitable set. Thevisualized LAMP were modified via optimize the temperature, the concentration ofdNTPs, MgSO4, FIP/BIP. Detection specificity were evaluated use the blood DNA ofP.v., P.f., P.m. and P.o. as templates. Diagnostic efficiency was evaluated use bloodand blood filter samples treated differently as template. Detection sensitivity wereevaluated use the series concentration of plasmid solution and parasitemia blood DNAas template. Results:8primer sets were generated and Pg-18S rRNA primers set were selected as the most suitable. The condition and content of modified visualizedLAMP system was carried out according to optimization，each20μL of reactionmixture contained the following:20mM Tris-HCl pH8.8,10mM KCl,10mM(NH4)2SO4,0.1%Tween20、8U Bst DNA polymerase,1.4mM dNTPs,9mM MgSO4,0.2μM F3/B3,2.5μM FIP/BIP,0.8μM LPF/LPB, template1.0μL. Then64℃react60min. The modified visualized LAMP can detect4species of Plasmodium specialty.It has good efficient in detecting samples treated differently. It can detect Plasmodium18s rRNA plasmid as low as3.1×100copy/μL, along with the threshold timedecreases correlated as plasmid concentration increased. It can detect seriesconcentration of parasitemia blood DNA as low as4.863p/μL.Part2Benefit-cost comparison between modified visualized LAMP withself-made reagents and commercially available Loopamp DNA amplificationkit in detecting PlasmodiumMethods: Modified visualized LAMP with self-made reagents and commerciallyavailable Loopamp DNA amplification kit were used to detect Plasmodiumrespectively. Detect efficiency, sensitivity and specificity between the two methodswere compared. Related costs and expenses between the two methods were alsocompared to estimate the usefulness of modified visualized LAMP to detectPlasmodium in field areas. Results: The two methods in detecting Plasmodium hadsimilar specificity. The modified visualized LAMP was more efficient and stabilized.The modified visualized LAMP can detect parasitemia of4.863p/μL which is10times of Loopamp DNA amplification kit sensitivity as48.63p/μL. The costs todetect single sample using modified visualized LAMP with self-made reagents wasabout7yuan/test, compared to the costs detecting using Loopamp DNA amplification kit which reached to166yuan/test. The former is about1/24of thelatter.Part3Evaluation of modified visualized LAMP in detecting malaria fieldsamplesMethods: The modified visualized LAMP was used to detect blood filter samplesfrom field. Results were compared with microscopy. Inconsistent samples wereanalyzed and re-checked using microscopy re-examination, nested PCR, DNAconcentration detect and case tracking survey, then evaluate the value of modifiedvisualized LAMP for field use during the malaria eliminating stage. Results: Within200filter blood samples of network reported cases collected form clinical detected bymodified visualized LAMP,186of which were Plasmodium positive and the14leftwere negative, with a coincidence rate of96.5%with microscopy. All inconsistentsamples were re-checked by nested PCR and got the same results with LAMP. Withinthe3LAMP and nested PCR positive but microscopy negative samples, according tore-examination by provincial microscope specialists,1was re-examined as P.v.positive with a low parasitemia of16p/μL,2as Plasmodium negative samples, whichafter tracking field case epidemic survey were confirmed to be imported P.f. cases.The two cases were anti-malaria treated before blood taken to make blood film.4samples detected negative by LAMP and nested PCR but positive by provincialmicroscope specialists re-examination were re-checked by DNA concentration detect,of which results showed there were DNA extracted in sample DNA solution. Conclusion1. To detect malaria parasites, a Plasmodium genus-specific18s rRNA primer setwas selected and a modified visualized LAMP method was established,thedetective sensitivity was4.863p/μL.2. Compared to commercially available Loopamp DNA amplification kit, themodified visualized LAMP with self-made reagents had similar specificity,higher efficiency and sensitivity, in addition, the cost was only about1/24ofLoopamp DNA amplification kit.3. Comparison of the diagnosis of field blood filter DNA samples by modifiedvisualized LAMP and microscopy examination, the former with highersensitivity, could be potentially used in detection cases with low parasitemia oranti-malaria drugs treated,which were difficult to be checked by routinemicroscopy.4. As a novel method for malaria parasites DNA detection, the modifiedvisualized LAMP is simple, highly efficient, sensitive, specific, nonexpensive,and suitable for large scale applications in the malaria eliminationstage of our country.