| Schistosomiasis is still one of the most important parasitic diseases worldwide, prevalent in 76 countries and regions of Asia, Africa and Latin America, and it is the major public health problem of the world. There are 6 major schistosome species, including Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium, Schistosoma mekongi, Schistosoma intercalatum and Schistosoma malay. In China, Schistosoma japonicum is the only causative species of schistosomiasis which may lead to hepatic periportal fibrosis and portal hypertension due to the deposition of a large number of Schistosoma japonicum eggs in tissues. Schistosomiasis japonica is not only severe disease, but also affect the economic development in schistosomiasis endemic areas. In 2004, schistosomiasis japonica as well as SARS and AIDS was listed into Class B of the infectious diseases in China.Schistosomiasis japonica was ever widespread in Jiangsu, Zhejiang, Shanghai, Anhui, Jiangxi, Hunan, Hubei, Sichuan, Yunnan, Guangxi, Guangdong, Fujian and so on. In the early days of new China, it had a total area of 14.8 billion square meters of snail, a total of 11.6 million cases of infection and 1 million or more of the threatened population. After 60 years of comprehensive prevention, it was estimated that 365,770 cases of schistosomiasis at the end of 2009, decreased by 11.42% compared to those in 2008. A total of 77 acute cases were reported, including 2 cases of schistosomiasis mansoni outside transported, which increased by 35.09% compared to those in 2008. A total of 24,282 patients with advanced schistosomiasis were treated, increased by 14.42% compared to those in 2008. Now the existed national snail areas are about 372358.69hm2, including new snail area 879.42hm2. Although the epidemicity of schistosomiasis in China has been effectively controlled, in some places schistosomiasis reemerges in recent years because of biological, natural, social, economic factors, population migration and a great change in policy support so on. The quantities of patients in schistosomiasis endemic areas and the infected snails increased significantly and the distribution of infected snails expanded in some areas, where had already been blocked the transmission of schistosomiasis. Additionally, the imported cases of schistosomiasis also increased. These suggest that the control of schistosomiasis has a long way to go in China.The key to control schistosomiasis is establishing an effective diagnostic method. The detecting techniques need to be developed more sensitive and specific. Now we always use Kato-Katz method as golden standard to evaluate some other diagnosis methods, but search for eggs in stools can be negative until parasite lay eggs and eggs are released into the intestinal lumen which will be 25-26 days after infection with Schistosoma japonicum. This direct parasitological technique is low sensitivity which hinders the diagnosis of early infection or individuals with low infection, and limits its application in evaluating chemotherapy. Immunodiagnostic techniques are more sensitive and easy to perform, and become a common epidemiological tool for the screening of target populations in many schistosome-endemic areas, but antibody detection is lack of specificity.More recently, LAMP, a simple rapid sensitive detection method is established. It uses Bst DNA polymerase with strand displacement activity in less than an-hour amplification under isothermal conditions. This method of detection has been used to detect different pathogens, such as African trypanosomes, Babesia orientalis, Cryptosporidium, Plasmodium, Toxoplasma gondii and schistosomes.In this study, we established a loop-mediated isothermal amplification (LAMP) method to detect whole blood DNA samples with Schistosoma japonicum infection. Schistosoma japonicum genomic repeated DNA sequence SjR2 (2718 3019 bp, 836 1036 bp), SjSH7 (688 883 bp) and Sj-nonLTR1 (2173 2376 bp) were selected and amplified using 4 groups of specific primers designed according to the principles of LAMP. The sensitivity and specificity were compared among LAMP methods using 4 different groups of primers. And the reaction system of LAMP was optimized. Furthermore, the LAMP method was used to detect whole blood DNA samples in 18 rabbits with Schistosoma japonicum infection at the different time points. We aimed at a 301-bp sequence of SjR2-1 to compare the sensitivity and specificity of PCR and LAMP assay in detecting Schistosoma japonicum derived DNA from experimentally infected rabbits and to explore the potential application of these two methods in the early diagnosis and evaluation for chemotherapy.The main results are as follows:1. Using different concentrations of betaine to optimize loop-mediated nucleic acid amplification, the results showed that the final concentration of 0.8M betaine could reach the optimal amplification efficiency. We tested the different amounts of betaine, such as the final concentrations of 1.6, 0.8, 0.4, 0.2 and 0 mol/L betaine, in LAMP reaction system, the positive control of DNA template (SjR2 2718 3019 bp segment) were all amplified, but the amplification buffer containing 0.8 mol/L Betaine was most efficient for LAMP.2. In the establishment of loop-mediated isothermal amplification (LAMP) system, our results showed that Triton X-100 had higher amplification efficiency than Tween-20. After the dilution, of the initial concentration with 1ng/mL the SjR2 2718 3019 bp purified PCR segment products ,we used the reaction buffer containing Tween-20 and Triton X-100 for LAMP amplification. The amplification sensitivity of Tween-20 was 10-3 ng / ml, and Triton X-100 was 10-4 ng / ml.3. Schistosoma japonicum genomic repeated DNA sequence SjR2 , SjSH7 and Sj-nonLTR1 were selected and amplified using 4 groups of specific primers designed according to the principles of LAMP. The sensitivity and specificity were compared among LAMP methods using 4 different groups of primers. The primers to Schistosoma japonicum SjR2(2718～3019 bp) could be specifically used to amplify the DNA segments from S. japonicum Chinese mainland strain, Philippine strain and Schistosoma mansoni genomic DNAs. The detection limit of S. japonicum Chinese mainland strain was 10-4 ng DNA.4. Amplified three groups of rabbit genomic DNA extracted from whole blood in the early stages of Schistosoma japonicum infection (1 week after infection) by LAMP and PCR , the results indicate that LAMP was more efficient than itional PCR in the early diagnosis of Schistosoma japonicum infection. 3 groups of different infected degrees of rabbits whole blood samples(1 week after infection) for LAMP detectng, either 200 or 1500 cercariae infection, the positive rate was 100%; however,the results of PCR showed that: the positive rate of 200 cercariae infection was 50%;the group of 1500 cercariae was 66.7% .The positive rate for PCR was increased with the rise of the Schistosoma japonicum infection degree. Thus, for SjR2-1 zone, it was better performance LAMP than PCR in the early diagnosis.5. Amplified all of three groups of rabbit whole blood samples before and after treatment of Schistosoma japonicum infection by LAMP and PCR , the dynamic results indicate that :for more sensitivity, the transfer negative time and negative conversion rate of LAMP were later and lower than PCR after drug treatment. The lowest positive rate of LAMP dropped to50% at 21 weeks after infection when sacrificed,but no later than 15 weeks after infection by PCR.Because after the drug treatment to eliminate parasites, specificity DNA fragments of Schistosoma japonicum remains a long time in peripheral blood of host (4-5 months or longer), so LAMP method may not be suitable for early evaluation of treatment assessment. 6. As a technical exploration, the preliminary results of LAMP used in the scene did not achieve the goals and needed further study. Detected 32 human whole blood samples collected in Jiangxi with fecal miracidia hatching positive by LAMP and PCR , the positive rate of LAMP was 37.5%, while PCR could not detected.The result also suggests that LAMP had high sensitivity. On the crowd of endemic area was not as good as lab tests and stability, and interpretation of results still needs to be clear.In summary, three sections of DNA repeat sequences of Schistosoma japonicum were choose and four groups of primers were designed to screen out by high sensitivity and specificity in this study. We establish the detection of Schistosoma japonicum infected rabbits in the Whole Blood Genomic DNA of the LAMP method. LAMP primers were used to select the best technology and general PCR detection of S. japonicum infection in the positive rates and negative rate after treatment, compared to evaluate the efficacy of early diagnosis and assessment of the value. LAMP was detected earlier than PCR, with the early diagnosis of schistosomiasis infection, but the LAMP method is not suitable for early evaluation of assessment . Initial application of LAMP assay samples crowd in endemic area, the results did not achieve the desired goals, still needs further study. |
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