Blastocystis spp. is a common intestinal protozoan parasites found in humans and many animals (Mei et al., 2022). The infection rate of Blastocystis spp. is about 0.5%~57% in developed countries and 30%~60% in developing countries (Zhang et al., 2019). More than 12 provinces in China have reported Blastocystis spp. infection, with a total infection rate of 3.37%. Among them, Fujian Province had the lowest prevalence (0.80%) and Guangdong Province had the highest prevalence (100%) (Deng et al., 2019). Several studies have reported that Blastocystis spp. can lead to clinical symptoms such as abdominal pain, diarrhea, vomiting, constipation, and irritable bowel syndrome (IBS) (Légeret et al., 2020; Asghari et al., 2021). In addition, patients with ulcerative colitis develop hematochezia after infection with Blastocystis spp (Unalan-Altintop et al., 2022). Rapid and accurate diagnosis of Blastocystis spp. infection is an important step in the prevention and control of blastocystosis, which plays an essential role in public health (Liu et al., 2023). However, the clinical manifestations of blastocystosis are non-specific and similar to those caused by other gastrointestinal diseases such as irritable bowel syndrome. Microscopic examination is the main method of clinical diagnosis for Blastocystis spp (McHardy et al., 2014)., which is prone to false negative results. Therefore, establishing a rapid, accurate, and convenient detection method for Blastocystis spp. has great significance.

Molecular biological diagnosis has great advantages in specificity and sensitivity, and is one of the commonly used diagnostic methods for blastocystosis. Among them, conventional PCR (cPCR) has been widely used in the diagnosis and genotyping of Blastocystis spp (Yoshikawa et al., 2004; Stensvold et al., 2006; Domínguez-Márquez et al., 2009; Khademvatan et al., 2018). However, specialized instruments and equipment are required for cPCR, including precise thermal cycling instruments, personnel trained in the technology, and maintaining a cold chain to preserve the heat-unstable reagents. The detection is usually completed in specialized testing centers, which are typically located far away from the high-risk population of infection. In contrast, field detection has certain limitations and has not yet been popularized. Therefore, cPCR detection is usually only performed in the laboratory and rarely used in the initial clinical diagnosis. A more rapid, simple, and accurate detection method is required.

In 2006, recombinase polymerase amplification (RPA) was developed in the UK, providing an isothermal amplification technique for nucleic acids (Piepenburg et al., 2006). The amplification of double-stranded DNA is accomplished through a series of enzyme reactions without thermal or chemical denaturation. Moreover, The results of RPA can be displayed by a lateral flow dipstick (LFD), which is suitable for detection in resource-poor areas or field areas (Wang et al., 2022). RPA-LFD overcomes the need for precision thermal cycling instruments and greatly improves detection efficiency while preserving high sensitivity, thereby allowing rapid visualization of the results (Piepenburg et al., 2006). In recent years, there have been several reports describing the successful application of RPA technology in different parasite detection. including Giardia duodenalis (Crannell et al., 2015), Entamoeba histolytica (Nair et al., 2015) and Leishmania Viannia spp (Saldarriaga et al., 2016).. For Blastocystis spp., only RPA diagnostic methods have been reported (Mei et al., 2023b), it is regrettable that there is no more convenient and visual RPA-LFD diagnostic method at present.

The succinate dehydrogenase subunit A (SDHA) gene is a novel genetic marker utilized in the typing and evaluation of the genetic diversity of Blastocystis spp (Higuera et al., 2020).. In this study, an RPA-LFD method targeting the SDHA gene was developed to detect Blastocystis spp. rapidly and accurately. This method can be implemented for onsite testing in hospitals or areas with inadequate medical conditions. To our knowledge, this is the first study on the detection of Blastocystis spp. using the RPA-LFD technique.

A variety of intestinal pathogens, including viruses, bacteria and parasitic protozoa, could cause diarrhea, which is the second leading cause of death among children (Kotloff et al., 2013; Caner et al., 2020; dos Santos David et al., 2022; Kotloff, 2022). Among them, the cause of chronic diarrhea is increasingly thought to be caused by parasitic protozoa infection (Lima and Guerrant, 1992), such as Blastocystis spp., Giardia spp., Entamoeba spp. Among them, Blastocystis spp. is the most widely distributed protozoa (Kaewjai et al., 2023). Similar clinical symptoms and different treatment methods highlight the importance of establishing a rapid and accurate diagnostic method for Blastocystis spp.

In this study, a simple, rapid, sensitive, and accurate diagnostic method for Blastocystis spp. was achieved by RPA-LFD assay. This method resolved the low sensitivity limitations of microscopic observation and the requirement for expensive equipment for cPCR in remote areas. Furthermore, this method has high sensitivity, and the samples showed positive results when the DNA concentration of Blastocystis spp. was greater than 1 pg/μL. The species with the same colonization site or close relationship with the target pathogen were prone to false positives in molecular diagnosis. The RPA-LFD method established in this study was proved to have no cross-reaction with these non-target pathogens, indicating that the method has good specificity.

In contrast to PCR, which can be heated to avoid non-specific amplification caused by binding between primers, nucleic acid amplification achieved by RPA under constant temperature easily generates primer dimers. The formation of primer dimers not only affects the amplification efficiency but also may affect the test results in case of low or no template concentration (Özay and McCalla, 2021). To overcome this limitation, several RPA primers and probes were designed with the SDHA gene as the target sequence. Five pairs of primers with higher amplification efficiency were screened by RPA assay. Subsequently, two pairs of primers and probes passed negative testing. In the further sensitivity test, SDHA-nfo-P3/P3-F1/P3-R3 showed the highest sensitivity, which then passed the specificity test. Therefore, SDHA-nfo-P3/P3-F1/P3-R3 was finally determined to be the best combination primer and probe.

The RPA-LFD assay was effectively carried out at 25~45°C, indicating that the RPA-LFD assay had lower requirements for temperature conditions and a wider detection range, which was more convenient for the detection of Blastocystis spp (Abd El Wahed et al., 2015).. In addition, existing RPA methods require agarose gel electrophoresis to read the results (Mei et al., 2023b). In contrast, the RPA-LFD method established in this study can make a diagnosis faster, as the LFD can observe the T-line within 3 min. The accuracy of the method was further confirmed by the detection of clinical samples. However, the distribution of different subtypes of Blastocystis spp. is related to many factors such as geographical location and season. The clinical samples used in this study were from Xinxiang City, China. It was reported that there were 8 subtypes (ST1-ST7, ST12) in the Chinese, and only ST1, ST3, ST4, ST6 and ST7 were reported in Xinxiang City (Deng et al., 2019; Zhang et al., 2019; Mei et al., 2023a). Although only ST3, ST4 and ST7 were included in the 56 clinical samples tested here, the results of RPA-LFD were completely consistent with those of cPCR, indicating that this method has good clinical application potential. In future studies, clinical samples containing more subtypes of Blastocystis spp. can be tested to further ensure the accuracy and stability of the method.

In summary, a rapid detection method for Blastocystis spp. was developed using RPA technology, with the detection results being visualized by LFD. This method overcame the requirements of cPCR for thermal cycling instruments and agarose gel electrophoresis instruments. Moreover, amplification was effectively completed in the temperature range of 25~45°C. The detection time was also greatly shortened, and the results were observed by LFD after 3 min of RPA reaction. The lowest detection limit of DNA was 1 pg/μL, which was consistent with the cPCR. In addition, this method showed good specificity. This study provided a simple, rapid, and accurate method for the diagnosis of Blastocystis spp.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

The studies involving humans were approved by the Ethical Commission of the Xinxiang Medical University. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study. The manuscript presents research on animals that do not require ethical approval for their study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

XM: Funding acquisition, Methodology, Writing – original draft, Writing – review & editing. CS: Data curation, Formal analysis, Investigation, Methodology, Writing – original draft. JX: Formal analysis, Investigation, Writing – original draft. LJ: Data curation, Investigation, Writing – original draft. SZ: Data curation, Investigation, Writing – original draft. ZY: Formal analysis, Writing – original draft. TX: Conceptualization, Formal analysis, Writing – original draft. ZZ: Conceptualization, Writing – original draft. SW: Conceptualization, Funding acquisition, Resources, Writing – review & editing.