Development of a Recombinase Polymerase Amplification Assay for Schistosomiasis Japonica Diagnosis in the Experimental Mice and Domestic Goats
Although the prevalence of schistosomiasis japonica has declined gradually in China, more accurate and sensitive diagnostic methods are urgently needed for the prevention and control of this disease. Molecular diagnostic methods are advantageous in terms of sensitivity and specificity, but they are...
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Frontiers Media S.A.
2021
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oai:doaj.org-article:70773c52693549d9bac137d6943731882021-11-16T06:31:10ZDevelopment of a Recombinase Polymerase Amplification Assay for Schistosomiasis Japonica Diagnosis in the Experimental Mice and Domestic Goats2235-298810.3389/fcimb.2021.791997https://doaj.org/article/70773c52693549d9bac137d6943731882021-11-01T00:00:00Zhttps://www.frontiersin.org/articles/10.3389/fcimb.2021.791997/fullhttps://doaj.org/toc/2235-2988Although the prevalence of schistosomiasis japonica has declined gradually in China, more accurate and sensitive diagnostic methods are urgently needed for the prevention and control of this disease. Molecular diagnostic methods are advantageous in terms of sensitivity and specificity, but they are time-consuming and require expensive instruments and skilled personnel, which limits their application in low-resource settings. In this study, an isothermal DNA amplification assay and recombinase polymerase amplification (RPA) combined with lateral flow dipstick (LFD) were set up. It was used to detect S. japonicum infections in experimental mice and domestic goats by amplifying a specific DNA fragment of S. japonicum. The lower limit of detection for the LFD-RPA assay was evaluated using dilutions of plasmid containing the target sequence. Cross-reactivity was evaluated using genomic DNA from eight other parasites. The effectiveness of the LFD-RPA assay was verified by assessing 36 positive plasma samples and 36 negative plasma samples from mice. The LFD-RPA assay and real-time PCR were also used to assess 48 schistosomiasis japonica-positive plasma samples and 53 negative plasma samples from goats. The LFD-RPA assay could detect 2.6 femtogram (fg) of S. japonicum target DNA (~39 fg genomic DNA of S. japonicum), only 10-fold less sensitive than real-time PCR assay. There was no cross-reactivity with DNA from the other eight parasites, such as Haemonchus contortus and Spirometra. The whole amplification process could be completed within 15 min at 39°C, and the results can be observed easily using the LFD. The sensitivity and specificity of the LFD-RPA assay were 97.22% (35/36, 95% CI, 85.47%–99.93%) and 100% (36/36, 95% CI, 90.26%–100%) in mice, and 93.75% (45/48, 95% CI, 82.80%–98.69%) and 100% (53/53, 95% CI, 93.28%–100%) in goats. By comparison, the sensitivity and specificity of real-time PCR were 100% (36/36, 95% CI, 90.26%–100%) and 100% (36/36, 95% CI, 90.26%–100%) for mice, and 97.92% (47/48, 95% CI, 88.93%–99.95%) and 100% (53/53, 95% CI, 93.28%–100%) for goats. The LFD-RPA assay exhibits high sensitivity and specificity for the diagnosis of schistosomiasis japonica, and it is an alternative method for diagnosis schistosomiasis japonica in low resource setting.Qinghong GuoQinghong GuoKerou ZhouKerou ZhouCheng ChenCheng ChenYongcheng YueYongcheng YueZheng ShangZheng ShangKeke ZhouKeke ZhouZhiqiang FuZhiqiang FuJinming LiuJinming LiuJiaojiao LinJiaojiao LinChenyang XiaWenqiang TangXiaonan CongXuejun SunYang HongFrontiers Media S.A.articleschistosomiasis japonicarecombinase polymerase amplification (RPA)real-time PCRdiagnosisdomestic goatsMicrobiologyQR1-502ENFrontiers in Cellular and Infection Microbiology, Vol 11 (2021) |
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schistosomiasis japonica recombinase polymerase amplification (RPA) real-time PCR diagnosis domestic goats Microbiology QR1-502 |
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schistosomiasis japonica recombinase polymerase amplification (RPA) real-time PCR diagnosis domestic goats Microbiology QR1-502 Qinghong Guo Qinghong Guo Kerou Zhou Kerou Zhou Cheng Chen Cheng Chen Yongcheng Yue Yongcheng Yue Zheng Shang Zheng Shang Keke Zhou Keke Zhou Zhiqiang Fu Zhiqiang Fu Jinming Liu Jinming Liu Jiaojiao Lin Jiaojiao Lin Chenyang Xia Wenqiang Tang Xiaonan Cong Xuejun Sun Yang Hong Development of a Recombinase Polymerase Amplification Assay for Schistosomiasis Japonica Diagnosis in the Experimental Mice and Domestic Goats |
| description |
Although the prevalence of schistosomiasis japonica has declined gradually in China, more accurate and sensitive diagnostic methods are urgently needed for the prevention and control of this disease. Molecular diagnostic methods are advantageous in terms of sensitivity and specificity, but they are time-consuming and require expensive instruments and skilled personnel, which limits their application in low-resource settings. In this study, an isothermal DNA amplification assay and recombinase polymerase amplification (RPA) combined with lateral flow dipstick (LFD) were set up. It was used to detect S. japonicum infections in experimental mice and domestic goats by amplifying a specific DNA fragment of S. japonicum. The lower limit of detection for the LFD-RPA assay was evaluated using dilutions of plasmid containing the target sequence. Cross-reactivity was evaluated using genomic DNA from eight other parasites. The effectiveness of the LFD-RPA assay was verified by assessing 36 positive plasma samples and 36 negative plasma samples from mice. The LFD-RPA assay and real-time PCR were also used to assess 48 schistosomiasis japonica-positive plasma samples and 53 negative plasma samples from goats. The LFD-RPA assay could detect 2.6 femtogram (fg) of S. japonicum target DNA (~39 fg genomic DNA of S. japonicum), only 10-fold less sensitive than real-time PCR assay. There was no cross-reactivity with DNA from the other eight parasites, such as Haemonchus contortus and Spirometra. The whole amplification process could be completed within 15 min at 39°C, and the results can be observed easily using the LFD. The sensitivity and specificity of the LFD-RPA assay were 97.22% (35/36, 95% CI, 85.47%–99.93%) and 100% (36/36, 95% CI, 90.26%–100%) in mice, and 93.75% (45/48, 95% CI, 82.80%–98.69%) and 100% (53/53, 95% CI, 93.28%–100%) in goats. By comparison, the sensitivity and specificity of real-time PCR were 100% (36/36, 95% CI, 90.26%–100%) and 100% (36/36, 95% CI, 90.26%–100%) for mice, and 97.92% (47/48, 95% CI, 88.93%–99.95%) and 100% (53/53, 95% CI, 93.28%–100%) for goats. The LFD-RPA assay exhibits high sensitivity and specificity for the diagnosis of schistosomiasis japonica, and it is an alternative method for diagnosis schistosomiasis japonica in low resource setting. |
| format |
article |
| author |
Qinghong Guo Qinghong Guo Kerou Zhou Kerou Zhou Cheng Chen Cheng Chen Yongcheng Yue Yongcheng Yue Zheng Shang Zheng Shang Keke Zhou Keke Zhou Zhiqiang Fu Zhiqiang Fu Jinming Liu Jinming Liu Jiaojiao Lin Jiaojiao Lin Chenyang Xia Wenqiang Tang Xiaonan Cong Xuejun Sun Yang Hong |
| author_facet |
Qinghong Guo Qinghong Guo Kerou Zhou Kerou Zhou Cheng Chen Cheng Chen Yongcheng Yue Yongcheng Yue Zheng Shang Zheng Shang Keke Zhou Keke Zhou Zhiqiang Fu Zhiqiang Fu Jinming Liu Jinming Liu Jiaojiao Lin Jiaojiao Lin Chenyang Xia Wenqiang Tang Xiaonan Cong Xuejun Sun Yang Hong |
| author_sort |
Qinghong Guo |
| title |
Development of a Recombinase Polymerase Amplification Assay for Schistosomiasis Japonica Diagnosis in the Experimental Mice and Domestic Goats |
| title_short |
Development of a Recombinase Polymerase Amplification Assay for Schistosomiasis Japonica Diagnosis in the Experimental Mice and Domestic Goats |
| title_full |
Development of a Recombinase Polymerase Amplification Assay for Schistosomiasis Japonica Diagnosis in the Experimental Mice and Domestic Goats |
| title_fullStr |
Development of a Recombinase Polymerase Amplification Assay for Schistosomiasis Japonica Diagnosis in the Experimental Mice and Domestic Goats |
| title_full_unstemmed |
Development of a Recombinase Polymerase Amplification Assay for Schistosomiasis Japonica Diagnosis in the Experimental Mice and Domestic Goats |
| title_sort |
development of a recombinase polymerase amplification assay for schistosomiasis japonica diagnosis in the experimental mice and domestic goats |
| publisher |
Frontiers Media S.A. |
| publishDate |
2021 |
| url |
https://doaj.org/article/70773c52693549d9bac137d694373188 |
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