A rapid and highly sensitive multiple detection of human adenovirus type 3, type 7 and respiratory syncytial virus by recombinase‐aided reverse transcription PCR

Abstract Background Polymerase chain reaction (PCR) has been widely used for many pathogen detection. However, PCR technology still suffers from long detection time and insufficient sensitivity. Recombinase‐aided amplification (RAA) is a powerful nucleic acid detection tool with high sensitivity and amplification efficiency, but its complex probes and inability of multiplex detection hinder the further application of this technology. Methods In this study, we developed and validated the multiplex reverse transcription recombinase‐aided PCR (multiplex RT‐RAP) assay for human adenovirus 3 (HADV3), human adenovirus 7 (HADV7), and human respiratory syncytial virus (HRSV) within 1 h with Human RNaseP protein as a reference gene to monitor the whole process. Results Using recombinant plasmids, the sensitivity of multiplex RT‐RAP for the detection of HADV3, HADV7, and HRSV was 18, 3, and 18 copies per reaction, respectively. The multiplex RT‐RAP showed no cross‐reactivity with other respiratory viruses, demonstrating its good specificity. A total of 252 clinical specimens were tested by multiplex RT‐RAP and the results were found to be consistent with those of corresponding RT‐qPCR assays. After testing serial dilutions of selected positive specimens, the detection sensitivity of multiplex RT‐RAP was two to eightfold higher than that of corresponding RT‐qPCR. Conclusion We conclude the multiplex RT‐RAP is a robust, rapid, highly sensitive, and specific assay with the potential to be used in the screening of clinical samples with low viral load.


| INTRODUC TI ON
Human adenovirus (HADV) and human respiratory syncytial virus (HRSV) are DNA and RNA viruses, respectively, of which human adenovirus types 3 and 7 (HADV3 and HADV7) are the most important pathogens causing epidemics in many countries and regions, as well as causing lower respiratory tract infections and even death. [1][2][3][4] Respiratory infections with HRSV can lead to bronchitis, bronchopneumonia, severe pneumonia, etc. In addition, studies have shown that HRSV is a major cause of pneumonia and bronchiolitis in infants. [5][6][7] The establishment of rapid and sensitive detection methods for these viruses is of great importance for the prevention and treatment of diseases. The main diagnostic methods for infectious diseases are nucleic acid amplification technology and immunological methods, PCR as the gold standard for virus diagnosis has made a great contribution in the prevention and control of the epidemic. [8][9][10] However, PCR suffers from long detection time and insufficient sensitivity.
Recently, we reported a novel two-stage nucleic acid amplification technique recombinase-aided PCR (RAP) by combining recombinase -aided amplification (RAA) with qPCR in a single closed-tube and demonstrated that RAP revealed ultra-high sensitivity (detection limits for HADV3 and HADV7 can reach single copy) and specificity, and could detect nucleic acids in less than 1 h, superior to current conventional qPCR assays. 11 However, RAP has some limitations.
First, RAP involves adding RAA mix to the cap of the PCR tube to complete the first stage of the RAA reaction, and then centrifuging the completed RAA product and mix with the qPCR buffer, which is cumbersome and relatively unstable. In addition, our previous study only demonstrated the effectiveness of RAP in single-plex DNA detection, but not in RNA virus and multiplex applications. Finally, the detection of internal reference targets was not included in RAP.
To overcome the drawbacks of RAP in multiplex and RNA virus detection, in this study, we developed a multiplex reverse transcription recombinase-aided PCR (multiplex RT-RAP) assay by using HADV3, HADV7, and HRSV to examine the ability of RAP in multiplex detection of DNA and RNA viruses. The human-derived Human RNaseP protein p38 gene (HRPP) was added in this method to improve the authenticity and credibility of the assay results. A paraffin (docosane) was added as a barrier to separate the RAA mix from the quantitative PCR (qPCR) mix. The multiplex RT-RAP was further validated with clinical samples.

| Sample collection and nucleic acid extraction
The clinical specimens of the respiratory tract were obtained from the Hunan Provincial Center for Disease Control and the Capital Institute of Pediatrics. The types of clinical specimens contained pharyngeal swabs and alveolar lavage fluid. The total DNA and RNA from 200 μL of these clinical specimens were extracted by Viral RNA/DNA Isolation Kit (Tianlong) following the manufacturer's instructions. The nucleic acids were eluted in 50 μL of elution buffer into 1.5 mL EP tube and stored at −80°C refrigerator.

| Primer, probe, and plasmid construction design
Complete sequences of HRSV were downloaded from the Nation Center for Biotechnology Information database (NCBI: https:// www.ncbi.nlm.nih.gov/) and aligned using Vector NTI 11.5.1 to locate the highly conserved regions. RAA primers (outer primers of RAP) for HADV3, HADV7, HRSV, and HRPP were cited from previous studies in our laboratory. 4,5,12 The qPCR primers (inner primers) and probes of HRSV and HRPP were designed using Primer 6 software and further evaluated using Primer-Blast in the NCBI website.
The sequences of primers and probes are presented in Table 1. All of them were synthesized and purified by Shanghai Bioengineering.

| Strategy of multiple RT-RAP assay
The multiplex RT-RAP assay consists of two stages, the first stage is the RT-RAA reaction, which reacts at a constant temperature of 39°C, the second stage is the qPCR reaction, which undergoes three processes: denaturation, annealing, and extension ( Figure 1). Since RT-RAA mixes and PCR mixes are not compatible, physical isolation of the two mixes is required. Here, a barrier called docosane (melting point 45°C) is added to separate the two compartments.
The RT-RAA reaction is completed in the compartment above docosane. During qPCR pre-denaturation of the second stage, docosane melts into a liquid state with less density than the RT-RAA mixture, enabling RT-RAA mixture mixes with the qPCR mixture in the compartment below docosane to start the second-stage qPCR reaction, which is completed after 20 cycles. The whole multiple RT-RAP assay lasts for 1 h.

| Optimization of multiplex RT-RAP assay working conditions
The optimal reaction conditions such as the volume of docosane, annealing temperature of qPCR, reaction time of RT-RAA, and primer concentration of qPCR were explored. First, to ensure that the two mixes could be effectively isolated before the RT-RAA reaction was completed, we explored the isolation effect of adding docosane in volumes of 15, 20, 25, and 30 μL, respectively. In addition, we explored the optimal annealing temperature for each primer at a plasmid concentration of 10 4 copies per reaction. Next, we examined the time effect of RT-RAA reaction at 5, 10, 15, and 20 min using HADV7 as an example with the plasmid template concentration of 1000, 100, 10, and 1 copies/reaction, respectively. Finally, we optimized and adjusted the concentration of qPCR primers in the reactions.

RAP assay
The sensitivities of multiplex RT-RAP were measured with the serial dilutions of the HADV3, HADV7, and HRSV plasmids ranging from 10 0 to 10 5 copies/μL in nuclease-free water. Meanwhile, the speci-

| Comparing the clinical performance of qPCR and RT-RAA versus multiplex RT-RAP
The clinical samples were simultaneously tested by previously re-

| Multiple RT-RAP reaction condition optimization
The volume ratio of RT-RAA mix to PCR mix was previously determined to be 1:4. 11 Our results showed that an effective barrier could be formed when 30 μL of docosane was added (Figure 2A).
In addition, the annealing temperature of the PCR stage exhibited a significant influence on the amplification efficiency of the reaction. Too high annealing temperature will reduce the amplification efficiency, while too low will produce nonspecific amplification.
Our experimental results showed that the best amplification efficiency was achieved when the annealing temperature was 55°C, and at the same time no obvious nonspecific amplification oc-

| Sensitivity and specificity of multiple RT-RAP assay
The sensitivity of multiplex RT-RAP for simultaneous detection of HADV3, HADV7, and HRSV was determined by detecting serial dilutions of the plasmids. The sensitivity of the assay was determined by Probit regression analysis for HADV3, HADV7, and HRSV to be 18, 3, and 18 copies per reaction, respectively (Table 2)

TA B L E 2
Assay data used for calculating the detection limit of HADV3, HADV7, and HRSV.

Copies/reaction
No. of positive samples tested by the RAP assay for detection of HADV 3, HADV 7, and RSV   (Table 3), the HRPP reference genes were all positive in the detection of the specimens, serving as a reliable monitoring of the entire reaction. Finally, we randomly selected three clinical samples positive for each of the three viruses and serially diluted these samples followed by both RT-qPCR and multiplex RT-RAP assays. The results showed that the sensitivity of multiplex RT-RAP assay for HADV3 and HRSV was two to fourfold higher than that of RT-qPCR, while the sensitivity of HADV7 was four to eightfold higher. These results demonstrated that our multiplex RT-RAP assay has high sensitivity and specificity.

| DISCUSS ION
Since  In conclusion, the proposed closed-tube multiplex RT-RAP is a rapid, highly specific, and highly sensitive nucleic acid detection platform, which has great potential to be used in the detection of various pathogens.

AUTH O R CO NTR I B UTI O N S
XM and XS designed the study. GF, RZ, FT, XS, MZ, and FT performed the experiments. GF, RZ, and XH analyzed and interpreted the data. XM and GF wrote the study. All authors provided a critical review and approved the final article.

ACK N O WLE D G E M ENTS
We gratefully acknowledge the assistance from the National Institute for Viral Disease Control and Prevention (IVDC) and the Chinese Center for Disease Control and Prevention (CCDC).

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare that there is no conflict of interest in this work.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.