Application of DNA-based diagnostics in detection of schistosomal DNA in early infection and after drug treatment

Abstract

Background: Research is now focused on identification of sensitive and specific diagnostic tests for early identification of schistosomal infection and evaluation of chemotherapy in field situations in China.

Results: This study compared loop-mediated isothermal amplification (LAMP) with conventional PCR as DNA-based diagnostic techniques for the early detection of schistosomal DNA and the evaluation of chemotherapy. The results showed that both PCR and LAMP assays targeting a 301 base pair (bp) sequence of the highly repetitive retrotransposon, SjR2, amplified DNA from schistosomes but were unable to distinguish between schistosome species. LAMP and conventional PCR were shown to amplify the target sequence of the SjR2-pCR2.1 recombinant plasmid template with limits of detection of 10⁻⁴ ng and 10⁻² ng, respectively, thus demonstrating the superior sensitivity of the LAMP method. Schistosoma japonicum DNA was detected in all serum samples obtained from the three experimental groups at 1 week post-infection by LAMP assay, while the rate of detection by conventional PCR ranged from 50% to 66%. The potential application of PCR and LAMP assays for the evaluation of artesunate and praziquantel chemotherapy was investigated. PCR was shown to be less sensitive for detection of schistosomal DNA in drug-treated rabbit sera than the LAMP method.

Conclusions: The data presented here indicate that LAMP is suitable for the detection of early infection in the groups primarily infected with Schistosoma japonicum, such as migrants, travellers, military personnel and the younger age groups. However, it is less suitable for evaluation of the efficacy of chemotherapy in the early stages because of its high sensitivity.

Figure 1

Sensitivity of the LAMP and PCR methods for detection of recombinant plasmid of SjR2-pCR2.1. (A) The LAMP assay method showed the higher sensitivity (10^-4 ng) than the PCR method. (B) PCR method was just able to detect more than 10^-2 ng of plasmid DNA. M: DNA marker; 1-8: 1 ng-10^-7ng plasmid DNA; 9: negative control without plasmid DNA.

Figure 2

Specificity of the LAMP and PCR methods using genomic DNA from different species samples. (A) Specificity of LAMP method. (B) Specificity of PCR method. 1-10: The recombinant plasmid of SjR2-pCR2.1, Schistosoma mansoni, Schistosoma japonicum (Philippine strain), Schistosoma japonicum (Chinese mainland strain), Clonorchis sinensis, Oncomelania hupensis, Plasmodium vivax, Plasmodium falciparum, uninfected rabbits and negative controls.

Figure 3

Detection of early Schistosoma japonicum infection in rabbits by LAMP and PCR asay. (A) The LAMP assay detected all the blood samples at 1 week after Schistosoma japonicum infection. (B) PCR method just detected partial blood samples at 1 week post-infection.

Figure 4

Liver pathology with H&E staining in rabbits after artesunate or praziquantel treatment. (A) Representative image of the liver section of Group 1 (× 200); (B and C) representative image (× 200) of egg granuloma in the liver section of Group 2 and Group 3, respectively.

Figure 5

Dynamic detection of schistosomal DNA in infected rabbits following artesunate or praziquantel treatment. (A) Dynamic results by LAMP method. (B) Dynamic results by PCR method.

Figure 6

Experiment schedule of infection, drug treatment and blood collection.