Loop-mediated Isothermal Amplification (LAMP) For Early Diagnosis And Therapy Evaluation Of Schistosoma Japonicum Infection

In our previous studies, nucleic acid detection assays have been proved a potential tool for the early detection and therapy evaluation of schistosomiasis japonica in rabbit models with moderate and heavy intensity infection. In this study, LAMP method was successfully established, and four groups of light-infection rabbit models, which was more consistent with the current epidemiological situation were used to evaluate the utility of LAMP for early detection and therapy evaluation of schistosomiasis. Meanwhile, LAMP assay was optimized. Then three established methods, such as common PCR, nested-PCR and LAMP were compared together to evaluate the ability of LAMP assay for detedction of S. japonicum infection and efficacy of chemotherapy. This study includs five parts:PartⅠDetection of S. japonicum DNA in serum of rabbit model by established LAMP assayOn the basis of PCR assay, LAMP method was established. The detectin limit was 1.02 copies, which was 10 times more sensitive than nested-PCR, and 100 times more sensitive than common PCR. No cross-reactivity was observed with S. mansoni, C. sinensis and T. spiralis. For early detection and therapy evaluation in a rabbit model infected with 500 S. japonicum cercariae, LAMP could also amplify the specific DNA fragment from the serum at the 3rd day post-infection, and the DNA detection became negative at the 17th week post-treatment, which was consistent with nested-PCR, but was 1 week later than PCR assay, due to its higher sensitivity. However, LAMP assay was easy to perform, reacted rapidly and was inexpensive, it may therefore be applied for field diagnosis in endemic area. PartⅡOptimization of LAMP reaction system and reaction coditionThe optimum reaction system of LAMP: In a total volum of 25μL, contains 1×Bst-DNA polymerase buffer, 4mmol/L MgSO4, 1.0mmol/L dNTP, 0.24mmol/L out primers, 0.96mmol/L inner primers, 0.8mol/L betaine, 8 U Bst-DNA polymerase, and 4μL template DNA. Traditional phenol-chloroform method was selected to extract DNA from serum samples, compared with other three DNA extraction kits. Observing the color change(from orange to green) is more accurate to identify the amplification by addition of SYBR GreenⅠafter incubation.PartⅢLAMP assay for early detection and therapy evaluation of light-infection schistosomiasis japonicumFour grades of light-infection rabbit models infected with 200, 100, 50 and 30 S. japonicum cercariae were established, to evaluate the utility of nucleic acid amplification methods for field diagnosis, especially for LAMP assay. The results showed that all the three amplification methods, including common PCR, nested PCR and LAMP assay, could detect S. japonicum DNA at the 3rd day post-infection in four different groups of light-infected rabbit model, especially for the rabbit infected with 30 S. japonicum cercariae which EPG was 14. But LAMP assay was the most rapid method, and the result was easier to observe than the other to PCR methods. For the therapy evaluation, LAMP detection became negative at 14 weeks post-treatment (21 weeks post-infection) in groupⅠand groupⅡ, which was consistent with nested-PCR assay, but was 2 weeks later than common PCR. In groupⅢand groupⅣ, LAMP and nested-PCR detection became negative at 10 weeks post-treatment (17 weeks post-infection), which was one week longer than common PCR, indicating a higher sensitivity of LAMP than the PCR method. The results showed chemotheraputic efficacy was correlated with the infection intensity. LAMP assay had a higher sensitivity than common PCR, and was more rapid than nested-PCR, had a potential for field diagnosis in endemic area.PartⅣApplication of LAMP for clinical diagnosis of schistosomiasis japonicum and for evaluating chemotheraputic efficacyAfter getting the encouraging results from animal model, LAMP was used to detect 110 sera samples of patient, the results showed that the positive rate(95.5%) of LAMP was higher than common PCR(87.3%) and nested-PCR(89.1%). For 45 sera of healthy persons from non-endemic area, no positive result was found by all the three nucleic acid amplification methods. The specificity of the three assays was 100%. The negative predictive value(NPV) of common PCR, nested-PCR and LAMP was 87.5%, 88.9%, 95%, respectively. While the positive predictive value(PPV) of the three methods was 93.2%, 91.6%, 91.3%, respectively. And for 47 sera of patient after 3 months, 6months and 9 months post-treatment, the negative rate of LAMP was 23.4%, 61.7%, 83.0%, respectively. And the negative rate of PCR was 31.2%, 68.1%, 89.4%, respectively, and was 27.7%, 76.6%, 85.1% for nested- PCR. For the two immunologic methods(ELISA and IHA), the negative rate was 17.0%, 19.1%, 25.5% and 23.4%, 42.5%, 31.9%, respectively. The difference between nucleic acid detection methods and immunological assays was significant, P<0.05.PartⅤEvaluation of LAMP for detection of different intensity infection of schistosomiasis in patient and for evaluation of chematheraputic efficacyTo evaluate the clinical utility of LAMP for diagnosis of patients and for therapy evaluation, 110 sera of patinets with chronic disease and 47 sera of patients post-treatment were classfied according to the level of EPG. The detection results showed that the possitive detection rate of LAMP for heavy intensity infection patients was 100%(4/4), for moderate intensity infection patients was 92.3%(12/13), and for light intensity infection patients was 95.7%(89/93). The positive rate of common PCR was100%(4/4), 84.6%(11/13), 86.0%(80/93), respectively. And was 100%(4/4), 100%(13/13), 87.1%(81/93) for nested-PCR. While for two immunological methods(ELISA and IHA), the positive rate was 100%, 84.6%, 83.9% and 100%, 92.3%, 91.4%, respectively. The detection rate of three nuleic acid amplification method and two immunological methods was correlated with the intensity of infection. For therapy evaluation, the negative rate for heavr, moderate and light intensity infection of LAMP in sera at 9 months post-treatment was 100%,100% and 79.5%, respectively. Common PCR was 100%, 100% and 87.2%, respectively. And was 100%, 100%, 82.1% for nested-PCR. The negative rate of ELISA and IHA maintained at a low level, ranged from 0%~28.2% and 0%~33.3%, respectively. The difference between nucleic acid detection methods and immunological assays was significant, P<0.05. The negative rate was correlated with infection intensity and the duration post-treatment.LAMP assay had a similar sensitivity with nested-PCR, and was higher than common PCR assay. The consistency of LAMP for therapy evaluation with nested-PCR and common PCR was well,kappa index was 0.604 and0.735, respectively. But LAMP has several advantages over conventional PCR and nested-PCR assays. LAMP is more rapid and simple, does not require sophisticated equipment, only a water bath can accomplish the whole reaction process, and the amplification can be visually observed, which is of great value for applications in poorly equipped laboratories or in large-scale epidemiological studies conducted in isolated areas.