Hepatitis B virus (HBV) belongs to the hepatophilia deoxyribonucleic acid (DNA) viridae and can be divided into positive hepatophilia DNA virus and poultry hepatophilia DNA virus. In the former, HBV is one of the main pathogens of human liver disease. Currently, HBV includes nine genotypes (A∼I), as well as different subtypes of the respective genotypes, according to the phylogenetic analysis of whole genome sequence (Demosthenes et al., 2019). HBV can cause people to get hepatitis B. The main routes of its transmission include blood transmission, mother-to-child transmission, and medical-borne transmission (Li et al., 2021). The patients can appear with loss of appetite, nausea, upper abdominal discomfort, liver pain, fatigue, and other symptoms. After the HBV invades the human body, it gives rise to cellular immunity restriction, causing a series of continuous liver inflammatory reactions, resulting in the continuous aggravation of the immune injury of the body, which may eventually develop into cirrhosis and even into liver cancer (Nagaoki et al., 2021).

There are 70 million hepatitis B virus carriers in China, and every year, the number of new cases of hepatitis B is stable at about 1 million (Lu and Zhuang, 2009). The direct medical expenses for the treatment of hepatitis B and related liver diseases in China reach to 100 billion yuan every year. Furthermore, the blood transmission of HBV via contaminated blood products is of enormous threat in the case of emergency transfusions, especially in resource-limited regions, which are endemic for HBV. Therefore, hepatitis B is not only a burden in the field of social health economy but also a major public health problem that threatens the life and health of Chinese people. At present, the clinical application of anti-viral treatment can significantly improve the prognosis of patients. However, because the virus DNA in the nucleus cannot be completely removed, patients may have HBV reactivation phenomenon (Perrillo et al., 2015) and need to take medicine for life, and there is no effective radical cure. Therefore, an accurate, economical, and efficient pathogen diagnosis is the key to control the infection of HBV.

Presently, the detection method of HBV widely used in the clinic is fluorescence quantitative polymerase chain reaction (FQ-PCR), which has strict requirements to laboratory facilities and professionals. Thus, it is not suitable for popularization in a resource-limited area (Weber et al., 2019). Another method, enzyme-linked immunosorbent assay (ELISA) usually takes 1∼2 days to get the detection result, and the antibody preparation cycle is long (Ye et al., 2021).

In recent years, several isothermal technologies have been developed for rapid pathogen detection. Multienzyme isothermal rapid amplification (MIRA) technology, similar to recombinase polymerase amplification (RPA), is a novel rapid isothermal amplification technique of nucleic acid. This technology uses four core proteins, namely, recombinase-Rec A, DNA helicase-gp41, single-stranded binding (SSB) protein, and DNA polymerase-DNA pol I (Piepenburg et al., 2006). The helicase could cooperate with SSB to form the D-loop rapidly (Lu et al., 2021). The reaction can be completed within 5–30 min under the condition of 25°C–42°C, which can effectively avoid the disadvantages of the above isothermal amplification technology and is sensitive, specific, economic, and convenient. In addition, the lateral flow strip bands are easy to observe, and the results can be visually evaluated as positive or negative. This can also be done by the untrained personnel. Therefore, the interpretation of the lateral flow strip and the processing of the reaction itself require minimal equipment and no special trained operator (Liu et al., 2018; Miao et al., 2019). In addition, with the help of MIRA-LFD, the lateral flow strip can replace the other expensive equipment currently in use, to reduce the cost of each test. Thus, the detection of HBV by MIRA-LFD has the potential application in the developing countries with low-resource laboratories.

In order to solve the current problem of virus detection and promote the early detection and early treatment of hepatitis B in resource-limited rural area, our study combined MIRA and LFD technology to provide a rapid and easy to implement diagnosis method of HBV.

In this study, we designed specific primers in two highly conserved regions (region S and X) of the HBV genome and established an HBV DNA detection method via MIRA-LFD reaction. The entire amplification of MIRA-LFD only takes 10 min at 37°C, and then the dilution was added in the test trip and observed by the naked eye after 10 min. Compared with the existing method, this method is efficient and suitable to implement in the resource-limited area without the restriction of laboratory condition. In the MIRA-LFD reaction, the detection sensitivity can even reach 10 pg. Therefore, it is helpful to detect the potential HBV infection (Tu et al., 2017) and has the significant application in the prevention of the spread and infection of HBV.

With the rapid advancement of molecular biotechnology, several isothermal amplification techniques have been developed. Multiple cross displacement amplification (MCDA) (Wang et al., 2015), loop-mediated isothermal amplification (LAMP) (Nagamine et al., 2001; Wong et al., 2018), and cross-priming amplification (CPA) (Fang et al., 2009) can be carried out at about 60°C–65°C, and the amplification process usually ends within 60 min. In addition, the process of primer design is very complicated, and more than four primers need to be designed for amplification. Isothermal polymerase spiral reaction (PSR) is also performed at about 60°C for 60 min, and only one pair of primers need to be designed (Liu et al., 2015). Compared with the abovementioned methods, the RPA is mild and efficient. The reaction is performed at 37°C–42°C for 20–40 min and only needs to design one pair of primers (Piepenburg et al., 2006). However, it has been reported that RPA is not ideal for discriminating between mutations or identifying mutations based on the performance of a nested RPA-based assay (Ng et al., 2017).

The MIRA assay can be reacted continuously at 37°C–42°C with a reaction time of only 5–30 min. Additionally, it does not need the strict laboratory condition (Piepenburg et al., 2006). Therefore, it is appropriate in initial screening of infectious diseases. Lateral flow dipstick (LFD) technology is a high-sensitivity technique for the detection of trace antigens, which uses an amplified product containing a biotin to hybridize with the probe labeled 5-carboxyfluorescein (FAM) and bind to colloidal gold-labeled antibodies, to obtain intuitive detection results in a short time. Now, combined with other isothermal technologies, it has exhibited a great potential in rapid pathogen detection (Zeng et al., 2019) (Figure 7).

There are still several potential limitations in the present study. First, due to the numerous subtypes of hepatitis B virus, the primer sequence can only identify some of these subtypes, but not all of them. The HBV subtypes that do not match the primer-binding regions cannot be detected. To some extent, it limits the wide usage of this method. Second, DNA extraction process is still required. Although some reagent, such as chelex-100, has simplified the DNA extraction process, the DNA extraction process is still a limitation of the entire process. Our method did not combine the DNA extraction and rapid amplification into one step. Third, the present method contains only one MIRA reaction and one LFD strip, which is difficult to detect the HBV with mutations in the primer recognition sequence. It is possible to add additional reaction and test strip for detecting other mutations in the future optimization of MIRA-LFD.

In this study, the combination of MIRA and LFD is a simple and feasible method without the restriction of special instrument. The entire detection of MIRA-LFD only takes 10 min at 37°C, and then the dilution was added in the test trip and observed by the naked eye after 10 min. This fast and easy-to-read-out method has the potential application in the developing countries with low-resource laboratories.