RESUMO
Objective To establish the method for extracting exogenous short DNA fragments of Schistosoma japonicum from urine samples, and to evaluate the efficiency of this method for extraction from urine samples treated with various methods. Methods The S. japonicum SjG28 gene fragment was selected as a target sequence, and the 81 bp short DNA fragment was amplified on the target sequence using PCR assay. Following characterization using sequencing, the short DNA fragment was added into the urine samples as an exogenous short DNA fragment. Primers and probes were designed with SjG28 as a target gene, to establish the real-time fluorescent quantitative PCR (qPCR) assay. The sensitivity of this qPCR assay was evaluated with exogenous short DNA fragments that were diluted at a 1:10 dilution ratio as the DNA template, and the specificity of the qPCR assay was evaluated with the genomic DNA of S. mansoni, S. haematobium, Babesia, Ancyiostoma duodenaie, Cionorchis sinensis, and Paragonimus westermani as DNA templates. Exogenous short DNA fragments were added into artificial and healthy volunteers’ urine samples, followed by pH adjustment, centrifugation and concentration, and the efficiency of extracting exogenous short DNA fragments from urine samples was compared with the QIAmp Viral RNA Mini Kit (Qiagen kit) and BIOG cfDNA easy kit (BIOG kit). Results An 81 bp small DNA fragment of S. japonicum was successfully prepared, and the lowest detection limit of the established qPCR assay was 100 copies/μL of the 81 bp small DNA fragment of S. japonicum. If the genomic DNA of S. japonicum, S. mansoni, S. haematobium, Babesia, A. duodenaie, C. sinensis, and P. westermani served as DNA templates, the qPCR assay only detected fluorescent signals with S. japonicum genomic DNA as the DNA template. If the pH values of artificial urine samples were adjusted to 5, 6, 7 and 8, the recovery rates were (49.12 ± 2.09)%, (84.52 ± 4.96)%, (89.38 ± 3.32)% and (87.82 ± 3.90)% for extracting the exogenous short DNA fragment of S. japonicum with the Qiagen kit, and were (2.30 ± 0.07)%, (8.11% ± 0.26)%, (13.35 ± 0.61)% and (20.82 ± 0.68)% with the BIOG kit, respectively (t = 38.702, 26.955, 39.042 and 29.571; all P values < 0.01). If the Qiagen kit was used for extracting the exogenous short DNA fragment from artificial urine samples, the lowest recovery rate was seen from urine samples with a pH value of 5 (all P values < 0.05), and there were no significant differences in the recovery rate from urine samples with pH values of 6, 7 and 8 (all P values > 0.05). Following centrifugation of artificial [(64.30 ± 1.00)% vs. (58.87 ± 0.26)%; t = 12.033, P < 0.05] and healthy volunteers’ urine samples [(31 165 ± 1 017) copies/μL vs. (28 471 ± 818) copies/μL; t = 23.164, P < 0.05]. In addition, concentration of artificial urine samples with the 10 kDa Centrifugal Filter and concentration of healthy volunteers’ urine samples with the 100 kDa Centrifugal Filter were both effective to increase the recovery of the Qiagen kit for extracting the exogenous short DNA fragment of S. japonicum (both P values < 0.01). Conclusions A method for extracting exogenous short DNA fragments of S. japonicum from urine samples has been successfully established, and the Qiagen kit has a high extraction efficiency. Adjustment of urine pH to 6 to 8 and concentration of healthy volunteers’ urine samples with the 100 kDa Centrifugal Filter are both effective to increase the efficiency of extracting exogenous short DNA fragments of S. japonicum.
RESUMO
Objective To comprehensively evaluate the effects of indirect hemagglutination test(IHA),enzyme?linked im?munosorbent assay(ELISA),and dipstick dye method(DDIA)in the diagnosis of schistosomiasis japonica at different preva?lence by using Meta?analysis. Methods Through the literature review according to the inclusion and exclusion criteria,a data?base was established,and by using Meta?disc and R software,the Meta?analysis was performed including the threshold test,het?erogeneity test,weighted by the quantitative effect of merger,SROC curve fitting,etc. Results A total of 60 papers were in?cluded in the final analysis. The sensitivities of IHA were 0.84,0.76 and 0.94 in heavy,medium and low endemic areas,and specificities were 0.73,0.64 and 0.73 respectively;the sensitivities of ELISA were 0.88,0.80 and 0.93 in heavy,medium and low endemic areas,and the specificities were 0.59,0.59 and 0.62 respectively;the sensitivities of DDIA were 0.93,0.81 and 0.93 in the heavy,medium and low endemic areas,and specificities were 0.66,0.69 and 0.59 respectively. The weighted sensi?tivities of IHA,ELISA and DDIA were 0.83,0.87 and 0.90 respectively;the weighted specificities were 0.69,0.60 and 0.62 re?spectively. The areas under the curve of SROC were 0.89,0.96 and 0.92 in IHA,ELISA and DDIA respectively. Conclusions In different prevalence,the effectiveness of different methods for serological diagnosis of schistosomiasis is different. The sensi?tivity and specificity of all diagnostic methods of schistosomiasis need to further improve.