Enhancement of Recombinase-Aided Amplication (Raa) Assay by Betaine and Pullulan

In this research, nucleic acid amplication enhancers suitable for recombinase-aided amplication (RAA) assay were studied for the rst time, and amplication of a long-fragment (509 bp) by the RAA assay was initially explored. Using recombinant plasmids and clinical samples, RAA uorescence and basic methods were used to evaluate the ecacy. The uorescence method was evaluated by threshold time (TT) and uorescence value, and the basic method was interpreted by 2% agarose gel electrophoresis. Taking a previously established RAA assay for HPV18 as an example, we demonstrated that the addition of 0.2, 0.4, and 0.6 M betaine and 10% pullulan could enhance RAA. The new RAA assays with betaine and pullulan were called B-RAA and P-RAA, respectively. In the B-RAA and P-RAA uorescence methods, TT values could be shortened by 1.72–2.32 and 2.60 minutes, respectively, and uorescence values could be enhanced by 8847.25–9094.37 and 5250 mv, respectively. In the basic method, sensitivity could be increased by 10-fold. We successfully amplied a long-fragment of 509 bp in a P-RAA assay with a sensitivity of 10 2 copies/μL (compared with 10 3 copies/μL in the RAA assay). We thus conclude that betaine and pullulan are effective additives to enhance RAA assays.


Introduction
Recombinase-aided ampli cation (RAA) assay is a new technique for nucleic acid ampli cation under isothermal conditions that employs recombinase, single-stranded DNA binding (SSB), and DNA polymerase [1]. The procedure is usually conducted at 37℃-42℃. RAA combines recombinase obtained from bacteria or fungi with speci c primers to form an enzyme-primer complex. When this complex locates its complementary sequence on the template DNA, the template DNA is unlinked with the help of SSB, and a new complementary chain is formed under the action of DNA polymerase. The RAA method can achieve ampli cation within 20 min and, thus, presents the advantages of short reaction times and simple operation procedures over traditional PCR assays, as illustrated in previous reports on the detection of SARS-CoV-2 (e.g., Xue G et al. [2], Wu T et al. [3], and Wang J et al. [4]) and other pathogens [5][6][7][8][9].
Although RAA is a promising isothermal nucleic acid detection method, the primer and probe designs for this technique are more complex than those for PCR primers and probes because RAA requires primers and probes with greater lengths. The design of RAA primers and probes is closely related to the detection sensitivity and speci city of the assay. Utilization of degenerate bases or mismatches in the probe or primer sequence could reduce the sensitivity and increase the non-speci city of ampli cation. Another limitation of the RAA assay is the presence of relatively short amplicons (100-200 bp, referred to as shortsegment ampli cation in this paper). To address these issues, we attempted to explore some chemical additives to improve the sensitivity and speci city and overcome the amplicon size limitation of RAA assays.
We previously reported a conventional RAA uorescence assay for HPV 18 with a sensitivity of 10 copies/ µL [10]. Taking this assay as an example, in the present study, we explored two chemical additives, namely, betaine and pullulan polysaccharides, as potential enhancers for RAA. The new RAA methods added with these two enhancers are called B-RAA and P-RAA, respectively. We also examined the RAA of a ~ 500 bp template (referred to as long-fragment ampli cation in this paper) with the addition of these two enhancers.

Materials and reagents
The RAA probes and primers used for HPV18 detection are described in detail in our previous report [10] and listed in Table 1 In the RAA uorescence method, the RAA nucleic acid ampli cation kit ( uorescence method) was used to con gure a reaction system of 50 µL. The system consisted of 25 µL of reaction buffer, 2.1 µL of each forward and reverse primer (10 µM), 0.6 µL of the HPV18 probe (10 µM), 1 µL of 10 copies/µL plasmid template or DNase-free water (negative control), and 2.5 µL of 280 mM magnesium acetate. The reaction tube was incubated in an isothermal oscillatory homogenizer (RAA-B6100) at 39℃ for 4 min, brie y swayed and centrifuged, and then transferred to a constant-temperature nucleic acid ampli cation detector (RAA-F1620) for 30 min at 39.0℃. The results were observed in real-time.

Original RAA basic method
In the original RAA basic method, the RAA nucleic acid ampli cation kit (basic method) was used to con gure a reaction system of 50 µL. The system, operating, and reaction conditions are identical to those described above for the uorescence method, but no probe was added to the system. Agarose gel (Beijing Rocco Xinye Biotechnology Co., Ltd.) was stained with SYBR Green I nucleic acid dye (10000×, electrophoresis grade; Solarbio, Beijing, China). Next, 2 µL of 5× Green Gotaq Flexi Buffer (Promega, USA) was mixed with 8 µL of the product puri ed by DNA extraction (phenol:chloroform:isoamyl alcohol = 25:24:1; pH > 7.8) or DL2000TM DNA Marker (TaKaRa, Japan) for 2% agarose gel electrophoresis and visualized with the Gel DocTMXR + Imaging System (Bio-Rad, USA).

B-RAA basic method
The reaction was identical to the original RAA basic method. The addition of betaine was conducted as described for the B-RAA uorescence method.

P-RAA uorescence method
The reaction was identical to that of the original RAA uorescence method, except that different concentrations of pullulan (Sigma-Aldrich, USA) were added. The pullulan additive was prepared as follows. Exactly 0.1 g of pullulan was added to a centrifuge tube containing 1 mL of deionized water and placed in an ultrasonic microwave cleaner several times to accelerate dissolution and prepare a 10% (w/v) stock solution. The prepared pullulan stock solution was serially diluted 10 times to obtain solutions of 1-10%, and 1 µL was added to different unit tubes [11].

P-RAA basic method
The reaction was identical to the original RAA basic method. Pullulan was added as described for the P-RAA uorescence method.
2.5 Long-fragment (500 bp) ampli cation by the original RAA, B-RAA, and P-RAA assays Using the probe and reverse primers designed in our previous work [10], we redesigned a forward primer (Table 1) to increase the length of the ampli cation product to 509 bp. The ampli cation method was identical to that in B-RAA and P-RAA. In the RAA long-fragment (509 bp) uorescence and basic methods, we used the original RAA, B-RAA, and P-RAA assays to amplify 10 2 -10 4 copies/µL HPV 18 plasmids and repeated the experiments eight times. We added 0.4 M betaine to B-RAA and 10% pullulan to P-RAA.

B-RAA basic method
The ampli cation products of the RAA basic method were electrophoretic on an agarose gel. In the absence of betaine, no ampli cation bands were generated from 10 copies/µL HPV 18 plasmids. However, when the nal concentration of betaine was 0.2, 0.4, or 0.6 M, the ampli cation bands and a few nonspeci c bands were observed (Fig. 1B).

P-RAA uorescence method
We repeated the P-RAA uorescence method eight times. Compared with the original RAA uorescence method without pullulan addition, RAA uorescence with 10% pullulan could steadily enhance the RAA assays by shortening the TT and increasing the uorescence values. P-RAA could shorten the TT value by 2.60 min, enhance the uorescence value by 5250 mv (Fig. 1C, Table 2), and increase the amount of RAA ampli cation products.

P-RAA basic method
Products ampli ed by the RAA basic method were electrophoretic on an agarose gel. Without pullulan, no band was ampli ed in the presence of 10 copies/µL HPV 18 plasmids. At pullulan concentrations of 2%, 4%, 8%, and 10%, the target fragment was successfully ampli ed, and only a few non-speci c bands were generated. The ampli cation effects improved when 8% and 10% pullulan were applied. Therefore, the addition of pullulan to the RAA basic method can also increase the sensitivity of the technique by 10-fold and reduce non-speci c ampli cation (Fig. 1D).
3.3 Long-fragment (509 bp) ampli cation by original the RAA, B-RAA, and P-RAA assays As shown in Fig. 1E, the ampli cation sensitivity of the original RAA and B-RAA could reach 10 3 copies/µL, while the sensitivity of P-RAA could reach 10 2 copies/µL. Moreover, the uorescence values of the ampli cation results under different concentrations of the plasmid were higher than those of the two other RAA methods, and the TT value was shortened in P-RAA ( Table 3). The enhancement effect of B-RAA on long-fragment ampli cation was not obvious. The sensitivity of the basic RAA method was lower than that of the uorescence method. Ampli cation of 10 2 -10 4 copies/µL HPV 18 plasmids was achieved, and the product was subjected to 2% agarose gel electrophoresis. B-RAA revealed no ampli cation bands. In the original RAA and P-RAA assays, the electrophoretic results of 10 4 copies/µL plasmids showed an ampli cation product of 500 bp; some nonspeci c bands were also observed in P-RAA (Fig. 1F). The use of 10% pullulan achieved the best enhancement effect on long-fragment ampli cation.

Sample detection by the original RAA, B-RAA, and P-RAA assays
During short-segment ampli cation, all samples could be detected by the three methods, and the detection results of the three RAA methods were consistent with our previous results [10]. The uorescence values in B-RAA and P-RAA are higher than that in the original RAA, and the TT values in these assays were less than or equal to that in the original RAA in 85.71% (18/21) and 76.19% (16/21) of the tested samples, respectively. The uorescence enhancement and TT value shortening effects in B-RAA were greater than those in P-RAA (Table 4).  Table 5).
The results of sample detection were consistent with those of plasmid detection. Betaine showed excellent enhancement effects for short-segment ampli cation, whereas pullulan demonstrated favorable effects for long-segment ampli cation.

Discussion And Conclusions
A nucleic acid ampli cation enhancer is a reagent that can improve the e ciency, speci city, and yield of nucleic acid ampli cation. Several reports on PCR enhancers are available. For example, betaine has been observed to assist in the opening of double-stranded templates with high GC contents during denaturation by reducing the template sequence melting temperature [12,13]. Dimethyl sulfoxide (DMSO) reduces the need for interchain or intra-strand reannealing to improve the speci city and productivity of PCR reactions [13,14]. Glycerol [14], trehalose [15], and formamide [16,17] have also been reported to enhance PCR ampli cation.
Reports on ampli cation enhancers used in isothermal nucleic acid ampli cation are scarce. In the present work, we explored two nucleic acid ampli cation enhancers, namely, betaine and pullulan, and demonstrated their effectiveness as additives for RAA isothermal nucleic acid detection. Our results showed that, compared with the original RAA uorescence method, the two enhancers (i.e., B-RAA and P-RAA) could shorten the TT value, reduce the peak time, and decrease the detection time. Moreover, these enhancers could enhance the uorescence value and increase the number of ampli cation products.
Compared with that of the original RAA basic method, the sensitivity of B-RAA and P-RAA is 10-fold greater and their non-speci c ampli cation is lower. Our results also indicated that betaine has better ampli cation enhancement effects than pullulan in the RAA assay for HPV18 (i.e., short fragment, 251 bp).
Betaine can help DNA polymerase pass through some complex secondary structures smoothly, thereby eliminating the pause in the extension process caused by the formation of secondary structures [12][13][14]18]. Given the long length of RAA probes and primers, secondary structures within the primers, probes and DNA template may easily be formed by winding. We speculate that betaine relaxes these secondary structures, thereby facilitating RAA. Pullulan is a water-soluble microbial polysaccharide produced by Aureobasidium pullulans fermentation. Wang R et al. used pullulan as an accelerator in cross-priming ampli cation (CPA) and found that 1% pullulan could increase the reaction rate of CPA by approximately 7 min. Some researchers have speculated that the addition of pullulan to the nucleic acid ampli cation system can improve the thermal stability of the polymerase and maintain its high activity, thus improving ampli cation e ciency [11]. The RAA system includes two types of enzymes, namely, recombinant enzyme UvsX and DNA polymerase, and the stability and activity of these enzymes are critical for ampli cation. The enhancement effect in our P-RAA study demonstrates that pullulan may exert a certain protective effect on the conformation of the enzyme, thereby enhancing the stability of the enzyme and the extent of ampli cation [11].
In the conventional (original) RAA method, the length of the amplicon is favorable when in the range of 100-200 bp; because amplicon lengths greater than 300 bp are rarely reported, the application of RAA to amplicon enrichment for sequencing is seriously hindered. In the present study, we rst observed that two enhancers could enhance the ampli cation e ciency of RAA. Thus, we applied these enhancers to the ampli cation of long fragments (509 bp). Our data showed that pullulan could improve the ampli cation e ciency of long fragments in the RAA uorescence method. While betaine and pullulan can enhance the ampli cation of short fragments, the former does not play a signi cant role in the ampli cation of long fragments and may sometimes even inhibit RAA ampli cation. The speci c reasons behind this nding require further investigation. We thus recommend pullulan as an additive to RAA assays for the ampli cation of long fragments. However, during conventional long-fragment ampli cation, the sensitivity of P-RAA is not much greater compared with that of the original RAA assay. This may be since the sensitivity of the RAA basic method is 10-100 times lower than that of the RAA uorescence method, and the extension of such a long-fragment leads to such a result.
We tested various concentrations of trehalose, polyethylene glycol, DMSO, and bovine albumin as nucleic acid ampli cation enhancers for RAA assay in this study, and the improvements we observed were not as notable as those produced by betaine and pullulan (data not shown).
In summary, we found that two nucleic acid ampli cation enhancers, namely, betaine, and pullulan, are effective additives for RAA assays. If shorter fragments are to be ampli ed via the RAA method, 0.2, 0.4, or 0.6 M betaine may be recommended; if longer fragments are to be ampli ed, 10% pullulan is recommended. However, we only performed the RAA assay for HPV 18 as an example and did not carry out experiments to detect other pathogens. The concentration and amount of enhancers may need to be adjusted within a speci c range to avoid inhibitory effects. The optimal working concentrations of betaine or pullulan must be determined through further experiments when RAA assay is to be applied to detect other pathogens.