An improved nucleic acid sequence-based amplification method mediated by T4 gene 32 protein.

The uptake of Nucleic Acid Sequence-Based Amplification (NASBA) for point of care testing may be hindered by a complexity in the workflow due the requirement of a thermal denaturation step to initiate the cyclic isothermal amplification before the addition of the amplification enzymes. Despite reports of successful enhancement of other DNA and RNA amplification methods using DNA and RNA binding proteins, this has not been reported for NASBA. Here, three single-stranded binding proteins, RecA, Extreme Thermostable Single-stranded binding protein (ET SSB) and T4 gene gp32 protein (gp32), were incorporated in NASBA protocol and used for single pot, one-step NASBA at 41 °C. Indeed, all SSBs showed significantly improved amplifications compared with the 2-step process, but only gp32 showed no non-specific aberrant amplification, and slightly improved the time-to-positivity in comparison with the conventional NASBA. For synthetic HIV-1 RNA, gp32 was found to improve the time-to-positivity (ttp) by average of 13.6% of one-step NASBA and 6.7% of conventional NASBA for the detection of HIV-1 RNA, showing its potential for simplifying the workflow as desirable for point of care applications of NASBA.

Introduction

Nucleic acid amplification testing (NAAT) is used for screening and identification of a pathogen for the diagnosis of pathogen-based infections in the clinical, veterinary and agricultural sectors. Polymerase Chain Reaction (PCR) is the gold standard methodology widely employed in centralized diagnostic laboratories. Its operational requirement for thermocycling, however, imposes engineering challenges for the deployment of PCR in point of care testing [1]. Commercial products on the market demonstrate these challenges have been successfully overcome, but elegant alternatives eliminating the need for thermocycling by adopting isothermal amplification methods are rapidly gaining popularity. Different isothermal amplification methods have been developed and adopted for clinical diagnostics, demonstrating efficient and fast amplification at a constant temperature [2].

In PCR, the elevated temperature step denatures double-stranded DNA (dsDNA) as required for primer annealing and extension. Isothermal amplification technologies employ different strategies to achieve this, including enzymatic activities or primer design to access dsDNA. Generally, primer binding sites are the initiation sites for DNA polymerases with strand displacement activity (e.g. large fragment of Bsu, Bst, and E. coli DNA Polymerase I, or phi29 DNA polymerase) leading to dsDNA separation and extension yielding single-stranded DNA to initiate the isothermal amplification [1, 2]. Isothermal methods vary in the way initiation is facilitated, with strategies categorized as (i) strand invasion facilitated by recombinases and single-stranded binding protein (SSB) in Helicase Dependent Amplification (HDA) [3], Strand Invasion Based Amplification (SIBA) [4] and Recombinase Polymerase Amplification [5], (ii) thermodynamic invasion and primer annealing in Nucleic Acid Sequence Based Amplification (NASBA) [6], transcription-mediated amplification (TMA), Loop mediated isothermal amplification (LAMP) [7, 8], (iii) the use of nicking enzymes in Strand Displacement Amplification (SDA) [9] and Nicking Enzyme Amplification Reaction (NEAR) [10].

NASBA is primarily employed to directly amplify RNA targets and—using a molecular beacon for detection—it was demonstrated to be less prone to false positives in the presence of genomic DNA [11, 12] and faster than alternatives relying on reverse transcription of the RNA into DNA without the need for DNase I treatment. A schematic representation of NASBA amplification is provided in Fig 1. In conventional NASBA process, a pre-denaturation step of the RNA at 65 °C is required, followed by annealing of the primers to the corresponding RNA template and addition of NASBA enzyme cocktail: Avian myeloblastosis virus reverse transcriptase (AMV RT), RNase H, and T7 RNA polymerase to kickstart the amplification at 41 °C. Because T7 RNA polymerase is thermolabile, NASBA is executed as a 2-step process, adding the enzymes after the thermal primer annealing step [13]. Recognizing NASBA and TMA are less frequently used than PCR, the assays are performed many thousands to millions of times a day in platforms including bioMérieux Nuclisens® and Hologic Panther®. Compared to other isothermal amplification methods, such as LAMP or Recombinase Polymerase Amplification (RPA), the 2-step process could be considered inconvenient for high throughput or point of care operation as each additional processing step may present an operational and engineering challenge [14]. Procedural simplicity is critical reduce cost, prevent malfunction and achieve robust, reliable operation [1]. A recent review on enhancement strategies for isothermal amplifications concluded that effective additives used to eliminate the thermal denaturation step include helicase, recombinases, endonucleases, ionic liquids, betaine, proline and trehalose [15], and SSBs used for decreasing non-specific interactions and to prevent nucleic acid degradation. Here, the potential of SSBs to facilitate primer binding and eliminate the thermal annealing step in NASBA is reported as enhancement strategy, facilitating NASBA as a single step method for enhanced field deployability.

Fig 1

A schematic representation of conventional NASBA and SSB mediated NASBA.

The conventional NASBA protocol requires a hybridization step before the addition of thermolabile NASBA enzymes to initiate the amplification and signal acquisition measuring the fluorescence intensity of molecular beacon. In single stranded binding protein mediated NASBA, the thermal denaturation-hybrization step is circumvented, allowing truly isothermal, single pot NASBA. In the graphic representation of the amplification cycle, the solid lines represent DNA, the wavey lines RNA.

Results and discussion

A truly isothermal, single-step NASBA method is anticipated to enhance NASBA’s potential for adoption in portable diagnostic devices, as it eliminates the need for complex temperature and fluidic control, hence simplifying instrument and operational design. In addition to their role decreasing non-specific interactions [15], SSBs have been employed to facilitate strand invasion and initiate amplification in various isothermal amplification methods [16, 17]. This evidence suggests SSBs may also be employed to avoid the thermal pre-denaturation step in NASBA. Here, 3 commercially available SSBs, namely E. coli RecA, Extreme Thermostable Single-stranded binding protein (ET SSB), and T4 gene 32 protein (gp32) were examined to assess their ability to substitute the thermal pre-denaturation step and simplify NASBA method (Fig 1). A myriad of techniques has been developed for the detection and quantification of nucleic acid amplification products, ranging from simple colorimetric reactions for visual read-out to more complex electrochemical and optical sensors; a comprehensive review focusing on point of care analysis can be found elsewhere [18]. Here, we used the fluorescence signal from a molecular beacon for fluorescence detection.

To verify the hypothesis that SSBs could facilitate the conventional 2-step NASBA to take place as a single pot reaction, the SSBs were incorporated into NASBA amplification mixtures (LRB-5, Life Sciences Advanced Technologies LLC, FL, USA), and amplification rate and yield were evaluated. Briefly, the reaction mixture contained primers, target RNA, NASBA enzymes (LEM-5) and molecular beacon for detection, all in NASBA reaction buffer. SSBs were added to the amplification mix at amount of 2 μg of RecA, 80 ng ET SSB, or gp32 (100–320 ng/μL; 1.5–9.6 μM) per reaction respectively. Unless specified, single-step NASBA reactions were performed at 41 °C for the one-step amplification of the synthetic HIV-1 gag RNA, one of NASBAs commercial targets with its HIV-1 gag gene primer set (S1 Table in S1 File) without the heat denaturation step in a qPCR machine (Biorad CFX Connect) with fluorescence signal acquisition at 1 min interval for 60 minutes. The resulting graphs for ET SSB and RecA and gp32 are presented in Fig 2a–2c. Please note the recorded amplification times do not include the extra time required for the addition of the amplification enzymes after thermal annealing using the 2-step protocol. Attenuated amplification was observed for all one step NASBA reactions.

Fig 2

The effect of SSBs on NASBA of the synthetic HIV-1 RNA gene using (a) 80 μg ET SSB, (b) 2 μg RecA and (c) 1 μg gp32 of a sample containing 2 x 105 copies of synthetic HIV-1 RNA (~Ct 23) using the HIV-1 NASBA primer-probe set (S1 File).

All graphs show conventional 2-step NASBA (red), 1-step NASBA (black dash/dot), 1-step NASBA with SSB (blue dashed), and NTC in absence (solid black) and presence (solid blue) of SSB (n = 3).

Data in Fig 2. confirm SSBs can be used to improve the time-to-positivity (ttp) of one-step NASBA at 41 °C, with varying effect on the amplification kinetics and specificity. The addition of ET SSB resulted in an increase in amplification rate matching the conventional NASBA at high RNA target input (2 x 105 copies, ~ Ct value of 23). Interestingly, the fluorescence intensity for the no template control (NTC) was also increased, indicating either a loss in specificity or aberrant collateral interaction with molecular beacon and hence increased risk of false positives past 60 min. In the case of RecA a slight improvement in the amplification rate compared to one-step NASBA was found, but the amplification was attenuated compared with conventional NASBA. Similarly to ET SSB, a slight increase in the NTC signal was observed in the presence of RecA. This only occurred after extended amplification times and is unlikely to cause false positives unless incubation times > 60 min are used (data not shown), but it does indicate a decrease in specificity of the amplification. RecA has been demonstrated to enhance fidelity in multiplex PCR [19] and LAMP [20], RecA was not investigated further.

The addition of gp32 improved one-step NASBA (1 μg), with the ttp of 17.3 ± 0.36 min for the one-step reaction comparable to the 18 ±0.57 min ttp of conventional 2-step process. Importantly, no increase in NTC signal was observed, suggesting no adverse impact on specificity. To our best knowledge, gp32 has not been reported for NASBA but has been assigned multifaceted roles in various DNA and RNA amplifications. When incorporated in PCR, gp32 increased amplicon length, PCR yield [21] and DNA sequencing read length [22] and alleviated PCR inhibition [23]. In T4 bacteriophage DNA replication, gp32 binding of ssDNA was not sequence-specific [24-26] and it facilitates replication, recombination and repair by delivering unfolded DNA to the respective enzymes [24]. In RPA and SIBA [4, 5] gp32 was demonstrated to aid recombinases UvsX and UvsY facilitating primer annealing by invading dsDNA targets, enabling exponential DNA amplification. Besides, gp32 enhances polymerase and RT activities by preventing the formation of secondary structures of template ssDNA and RNA, respectively, allowing for doubling of the yield of in vitro transcripts by T7 RNA polymerase and a significant increase for reverse transcriptase [27]. More recently, gp32 has shown to assist strand invasion of primers at moderate temperatures (30–45 °C) and to facilitate the formation of an initiation site for DNA and RNA amplification [4, 16, 25]. Based on the different roles gp32 has played in amplification, we propose its benefits in NASBA may be multifaceted. Its ability to stabilize displaced ssDNA may improve primer annealing in NASBA by stabilizing the single-stranded cDNA formed by AMV RT.

Recognizing further investigation will be required to elucidate the mechanistic role(s) gp32 plays in facilitating the initial cyclic amplification step of dsDNA by T7 polymerase in NASBA, the presented results do demonstrate gp32 can facilitate one-step NASBA detection of HIV-1with slightly improved ttp in comparison with the 2-step protocol, eliminating the need for thermal pre-denaturation. The effect of gp32 concentration in the amplification reaction on ttp was investigated, with results summarized in Fig 3a. The optimum performance of gp32 was observed at concentrations between 3–4.5 μM (~100–150 ng/μL), with ttp faster than both conventional and no SSB one-step NASBA procedure, yet the ttp gradually increased at higher concentrations suggesting a concentration dependent effect. Using 3 μM of gp32 as the optimum for HIV-1 gag gene primer set, we observed similar improvement for various RNA inputs (Fig 3b). With mechanistic detail of gp32s effect in one-pot NASBA is still unclear, the attenuating effect of higher levels of gp32 should be investigated further. To the authors best knowledge, there are no literature reports of single step NASBA protocols, but anecdotal reports confirmed in our laboratory suggest the reliance on the thermal annealing step varies for primer/targets. The effect of gp32 was studied using the Acrometrix HIV-1 control, and a near-negligible improvement in ttp (from 29 min to 28.5 min) was found using this full length HIV-1 RNA (S1 File), a result that may have been caused by the low target concentration (100 copies/reaction). Improvements in ttp using gp32 were found for Avian influenza A H5N1 RNA, decreasing ttp from 38 to 31 min in presence of gp32, compared with a ttp of 28 min for the two-step process (unpublished results), confirming the performance enhancement with another primer set.

Fig 3

(a) Effect of gp32 protein on the ttp for HIV-1 using NASBA and HIV-1 primer set (S1 File) (n = 3). (a) Optimization of gp32 using 0, 3, 4.5, 7.2 and 9.6 μM (0, 100, 150, 240, 320 ng/μL) gp32. Sample contained ~1 x 105 copies of synthetic HIV-1 RNA; conventional 2-step NASBA included as reference, NTCs as 2-step (no gp32) and 1-step (with 4.5 μM gp32) (b) Comparison of NASBA carried out as conventional 2-step, one step in absence of SSB, and 1-step using 3 μM gp32 for HIV-1 RNA samples ranging from Ct 20 (~1.5 x 106 copies) to Ct 34 (~1.5 x 102 copies).

In conclusion, SSBs can be used to decrease the reliance on a thermal annealing step in NASBA, enhancing the procedural simplicity by facilitating one-pot NASBA. ET SSB, RecA and gp32 were evaluated, and gp32 was selected for further study as it provided faster amplification and no non-specific amplification. One-pot amplification of the HIV-1 gag gene mediated by 3 μM gp32 enabled a moderate ttp improvement in average of 6 min in one-step NASBA and 2.4 min in conventional NASBA (not accounting the manual addition of amplification enzymes after primer annealing). The effects were confirmed across VLP RNA input levels from Ct 20 to Ct 34, with improvement in amplification across the studied range. While mechanistic studies explaining the role of gp32 in one step NASBA, as well as to examine the effect of the target and primer are required, we believe the results obtained using gp32 can reposition NASBA in clinical diagnostics, particularly for tests executed outside the laboratory setting, as COVID-19 pandemic has demonstrated the need for fast, decentralized NAAT.

Material and methods

NASBA and purification of target RNA amplicon

The two target RNA samples used in the NASBA study were purified from synthetic viral-like particles (VLP) RNA. We packaged artificial sequence into MS2 capsid, which created the VLPs to serve as a model for carrying target sequence of interest in this study (HIV-1 gag gene Genbank AntiSense, strain HXB2, Accession #k03455, nt 1359–1499). The VLP was prepared as previously described [28]. RNA purification was performed using spin column based Macherey-Nagel NucleoSpin RNA Virus Mini kit (catalog Number 740956.50, Scientifix Pty Ltd, Victoria, Australia) as per the manufacturer’s protocol. The amplification process was performed using commercial NASBA reagent from Life Sciences Advanced Technologies, Inc. (St. Petersburg, FL, USA). Briefly, 15 μL reaction buffer mixture (LRB) consists of 40 mM Tris HCl (pH 8.5), 70 mM KCl, 12 mM MgCl2, 15% dimethyl sulfoxide, 5 mM dithiothreitol (DTT), 1 mM dNTP mixture, 2 mM ATP, CTP and UTP mixture, 1.5 mM GTP, 0.5 mM ITP, 0.1 μM molecular beacon probe, 0.2 μM of P1 and P2 primers (Integrated DNA Technologies, IL, USA) and purified RNA.

In conventional NASBA, the reaction mixture was preincubated at 65 °C for 2 min then lowered to 41 °C, amplification reaction was initiated by addition of 5 μL of enzyme cocktail (LEM) containing three enzymes, namely 6.4 U AMV Reverse Transcriptase (AMV-RT), 32 U T7 RNA polymerase, and 0.1 U ribonuclease H. NASBA reaction tubes were incubated at 41°C for 60 min with signal acquisition at 1 min interval. NASBA primers [29] and RNA target sequence are detailed in S1 Table in S1 File.

In SSB mediated NASBA, SSBs were added to the amplification mix in combination with the LEM cocktail at 2 μg of RecA (M0249, New England Biolabs (NEB)), 80 ng ET SSB (M2401S, NEB) or gp32 (M0300, NEB) at a concentration range between 50–320 ng/μL per reaction respectively. The amplification and signal acquisition were initiated and carried out at 41 °C for 90 min without the 65 °C melting step.

Nucleic acid extraction and qPCR quantitation

The RNA purification of VLP RNA and Thermo Scientific™ AcroMetrix™ HIV-1 Control were performed according to kit protocol yielding 50 μL of purified nucleic acid extract. The purified RNA was serially diluted linearly by 10-fold in RNase-Free Tris EDTA (pH 7.5) buffer, and the quantitation of the series was established using qRT-PCR using hydrolysis probe method (Bioline SensiFAST™ Probe No-ROX One-Step Kit, Bioline AUS, Sydney) with HIV-1 gag primer-probe set (S1 Table in S1 File). Subsequently, the calibrated nucleic acid extracts (S1 File) were transferred to an 8-well strip of 0.2 ml polypropylene tubes at stored under -80 °C until required.

S1 Table and information of VLP RNA quantification.

(DOCX)

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4 May 2021

PONE-D-21-08924

An Improved Nucleic Acid Based Sequence Amplification Method Mediated by T4 Gene 32 Protein

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Reviewer #1: The manuscript from Nai, Doeven and Guijt describes an improvement to NASBA reactions by inclusion of single-stranded binding protein, particularly T4 gp32. This would be a good benefit to improving usability and application of NASBA, but I have a few points that I think should be addressed before acceptance for publication.

1) As the authors describe, an initial denaturation step at 60-65C is typically done before adding NASBA enzymes and running the detection reaction. But the reason for this step is pretty fundamental, typically NASBA reactions do not work at all without it. Here the data show that the "one-step" protocol is actually just fine, a few minutes slower than the standard two-step but otherwise amplifying and detecting target well. This is an unusual result and I wonder if the simplicity of the used template or something else about the particular reaction being done here gives rise to an abnormal finding. If the key bottleneck for wider use of NASBA is this heat step, it seems from the data here that that step is already unnecessary. It would be good to investigate other primers/targets to demonstrate that the gp32 effect is not assay-specific, but it could also add to this point which is very unexpected to me.

2) The authors describe better usability of NASBA for point-of-care settings, but the reaction requires a molecular beacon and real-time fluorescence as well as extracted RNA. Could the detection be done more simply? Could the VLPs be used directly without extracting RNA? I also don't see the benefit of encapsulating a synthetic RNA then extracting it again, if a mock sample is desired here then a real mock sample should be used, spiking the VLPs into a swab or blood or whatever.

3) Some statistical analysis would help to show that the benefits/differences between the conditions are significant. The authors at one point compare 17.3 and 18 minutes, for example, and the data in Figure 3 would benefit from some significance determination. In Figure 3 the error bars in (a) seem extremely small compared to (b), the number of replicates is not listed and I find the difference odd.

4) As the authors note there are other isothermal methods being more widely used, RPA, LAMP, etc. What's the benefit of NASBA over those? Is it just that NASBA is an alternative, or does it offer something else? Sensitivity shown here is pretty moderate and I'd think any of the methods could pick up 150 copies in 30 minutes, and some without requiring fluorescence detection. Some commentary on why NASBA would be useful, even if it's just "as COVID has demonstrated we need as many methods as possible".

4) Minor points:

It would be good to show the molecular beacon binding the target in Figure 1. Also it's not very clear which strands are DNA and which are RNA as the images and labels are very small.

NASBA and TMA are of course not obscure, rarely-used methods but diagnostic workhorse methods performed by the many thousands or millions every day in platforms like the Hologic Panther. Would be good to note this, and that the 2-step temperature requirement isn't a problem for widespread use just for simple applications.

In the introduction the authors state that NASBA is "less prone to false positives and faster than alternatives relying on reverse transcription of the RNA into DNA." NASBA is only less prone to false positives with molecular beacon detection, certainly not if using an intercalating dye or other readout, and it of course requires reverse transcription of the RNA into DNA otherwise it doesn't work as the authors' own cartoon shows. This should be restated.

Reviewer #2: Title: An Improved Nucleic Acid Based Sequence Amplification Method Mediated by T4 Gene 32 Protein

Manuscript ID: PONE-D-21-08924

Authors: Yi Heng Nai*, Egan H. Doeven, and Rosanne M. Guijt

Submitted to PLOS ONE

This manuscript describes an improved nucleic acid sequence-based amplification (NASBA) using single-stranded binding protein (SSB), T4 gene protein 32 (T4gp32). In this strategy, three SSBs were employed to construct a modified NASBA capable of eliminating the initial denaturation step and thus amplifying target RNA molecule in a single pot at 41 ℃. The authors applied this strategy to amplify synthetic HIV-1 RNA molecules. The initial denaturation step in the traditional NASBA is needed to disrupt the complicated secondary structure of long genomic RNAs and the authors should validate the benefits of this improved NASBA technology by amplifying long genomic RNAs but not short synthetic RNAs which might rarely require the initial denaturation. Furthermore, this manuscript is not well-structured and contains a lot of errors. Therefore, I would not recommend acceptance of this manuscript. Some of my other comments are as follows.

Comment 1. Nucleic Acid Based Sequence Amplification’ should be corrected to ‘Nucleic Acid Sequence-Based Amplification (NASBA)’ throughout the overall manuscript.

Comment 2. Many abbreviations such as ttp and ET SSB are not defined.

Comment 3. The authors are recommended to provide standard deviation for Ct data in Figure 2.

Comment 4. The authors need to present the experimental data obtained from RT-qPCR conducted to confirm the concentrations of target RNA.

Comment 5. The authors need to provide relevant references

1) Related to false positives of the NASBA reaction. (Page3, Line 59)

2) Related to the thermolability of the T7 RNA polymerase. (Page 4, Line 65)

3) Related to the T4gp32 effects including the increase of the DNA sequencing read length and alleviation of the PCR inhibition. (Page 5, Line 106)

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Submitted filename: Review comments (PONE-D-21-08924).docx

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4 Nov 2021

Dear Editor,

Please find enclosed submission with our revised manuscript as per instructed in the decision letter.

1) Addressing reviewer comments can be found in the document - 'Response to Reviewers'.

2) A marked-up copy of manuscript that highlights changes made to the original version.

An unmarked version of your revised paper without tracked changes.

We look forward to receiving a favourable outcome on this resubmission.

Regards

Yi Heng Nai

Submitted filename: Response to reviewers (PONE-D-21-08924).docx

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20 Dec 2021

15 Feb 2022

Reviewer #1: I thank the authors for addressing the comments from the reviewers, the resulting updated version here is better but I still have a fundamental point that now if anything needs more attention. Also my intent from the first round was to get some of the points raised added into the manuscript, the authors seemed to disagree with me but I maintain that the things raised in the first review are still relevant, if no experiment is required just some discussion or text I fail to see how that is "out of scope of the current communication.

Response

We would like to apologise not having addressed detection in detail.

Action taken:

A sentence and reference on detection have now been included from line 92:

“A myriad of techniques has been developed for the detection and quantification of nucleic acid amplification products, ranges from simple colorimetric reactions for visual read-out to more complex electrochemical and optical sensors; a comprehensive review focusing on point of care analysis can be found elsewhere [18]. Here, we used the fluorescence signal from a molecular beacon for fluorescence detection.”

But more importantly, both I and reviewer #2 pointed out that the thermal denaturation/annealing step is generally considered required for NASBA/TMA to work well. The authors are claiming it is not when gp32 is present. That would be great, so in the assay shown matching the speed of the typical 2-step protocol just by including gp32 is a nice result...but it is very plainly a result of using this specific assay, as the 1-step, non-gp32 version works surprisingly well.

Response:

We agree that for HIV-1, the one step protocol worked surprisingly well, a phenomenon occasionally observed (but not reported) for NASBA amplifications. Unfortunately, we have been unable to identify “typical” attenuation for one step NASBA reactions for benchmarking purposes, as articles typically refer to the protocols developed in the 1990’s without presenting the attenuation experienced. We hypothesize it depends on the primers used as well as the formulation of the amplification buffer. A discussion of the observed amplification in single pot NASBA has now been included (see below under action taken).

The presented research shows that T4GP32 outperforms the other SSBs in specificity, not displaying non-specific amplification. We included a reference to recent review of approaches for enhancing isothermal amplification (Özay, B. and S.E. McCalla, A review of reaction enhancement strategies for isothermal nucleic acid amplification reactions. Sensors and Actuators Reports, 2021. 3: p. 100033). Please note this review confirms that SSBs so far have only been used to decrease non-specific binding, emphasizing the novelty of the use of gp32 in the primer annealing.

A possible explanation is that the reagents from LSAT may have been stabilised with trehalose -without our knowledge - which may have helped the one-pot reaction, as trehalose has been demonstrated to enhance isothermal amplification reactions (Mok, E., Wee, E., Wang, Y. et al. Comprehensive evaluation of molecular enhancers of the isothermal exponential amplification reaction. Sci Rep 6, 37837 (2016). We have noted that the precise formulation is no longer provided by LSAT. Seven years ago was listed as 40 mM TrisHCl, pH 8.5 @ 25ºC, 12 mM MgCl2, 70 mM KCl, 5 mM DTT, 15% Dimethyl Sulfoxide ---- 2 mM rA,C,UTP, 1.5 mM rGTP, 0.5 mM Inosine 5'-triphosphate, 1 mM dNTP. LSAT has not mentioned a change in formulation, but simply removed the composition from the public domain. It is normal in the industry to keep the exact formulation of the buffer as a trade secret.

The performance enhancement by trehalose described in the Sci Rep article were obtained at concentrations of 0.4 M, a value that is unrealistic to be present as “contamination”, discrediting the hypothesis trehalose entered the buffer with the stabilised enzymes.

Importantly, we have experimentally tested most of the additives listed in the newly cited review [Özay, B. and S.E. McCalla)] to facilitate the single pot reaction, identifying gp32 as the most promising candidate for NASBA. In that study, the amplification performance in the LSAT NASBA buffer was similar to that in the buffer composed using the ingredients listed seven years ago, hence we have no experimental evidence the LSAT formulation may have changed (nor do we have evidence it did not). The study did confirm an enhancement in amplification for the single pot reaction in presence of trehalose, however, the gp32 results reported here were superior.

Considering we used the LSAT buffer as indicated in the method section of manuscript, the performance enhancement was found for gp32 but not for other SSBs, irrespective if we used the LST buffer or made our own, we continue to believe it is important to communicate our findings on gp32 to allow other researchers to elucidate and benefit from its action(s) in NASBA and other isothermal amplification methods.

Action taken:

Added to line 104/104

“Attenuated amplification was observed for all one step NASBA reactions.”

We also included

“ To the authors best knowledge, there are no literature reports of single step NASBA protocols, but anecdotal reports confirmed in our laboratory suggest the reliance on the thermal annealing step varies for primer/targets.”

Above this discussion to illustrate that the observed one-pot amplification is not unique, and has been observed, but not reported by others.

And After line 76, the following text was added, including the abovementioned reference

“A recent review on enhancement strategies for isothermal amplifications concluded that effective additives used to eliminate the thermal denaturation step include helicase, recombinases, endonucleases, ionic liquids, betaine, proline and trehalose [15], and SSBs used for decreasing non-specific interactions and to prevent nucleic acid degradation.”

Additionally, we replaced “eliminate” with “decrease the reliance on” to moderate the concluding statement.

And added

“ While mechanistic studies explaining the role of gp32 in one step NASBA, as well as to examine the effect of the target and primer are required, ..” to the conclusion

The authors own data in the rebuttal shows another amplicon where the 1-step gives very poor signal and the gp32 addition does not rescue it to the level of the 2-step, let alone faster as they (unconvincingly from the limited data points) claim in the manuscript. I feel this proves my point, not theirs, and the gp32/1-step effect may indeed by an artifact of the assay used in the main body of the text. 2 assays are shown here, and the authors' claims are only true for 1 of them. Either more primer/sets assays should be shown where the gp32/1-step is indeed as good as the 2-step, or the authors should modify the claims in the manuscript to be more honest and in line with the data.

Response:

We would like to thank the reviewer for pointing this out, and we understand his/her position. We however, confirmed out findings with H1N1 and the HIV gag gene, and then upon request found a strongly reduced efficacy of T4GP32 in the low copy number full HIV sample, as indicated in the first revision. We agree with Reviewer 1 that, considering we obtained this undesirable result, we should alter the manuscript to discuss this result.

Action taken:

After line 147, the following text was added:

“The effect of gp32 was studied using the Acrometrix HIV-1 control, and a near-negligible A weak improvement in ttp (from 29 min to 28.5 min) was found using this full length HIV-1 RNA primer set (Supplementary Information), a result that may be due to the low target concentration (100 copies/reaction). This more modest effect could be caused by the low sample concentration or by the decreased dependence of the HIV-1 gag gene primer set on the thermal annealing step. Improvements in ttp using gp32 were confirmed for Avian influenza A H5N1 RNA, decreasing ttp from 38 to 31 min in presence of gp32, compared with a ttp of 28 min for the two step process (unpublished results). “

That in circumstances where avoiding heat denaturation is paramount to NASBA utilization, then gp32 may help, but there may be sacrifices in performance depending on the assay. That is obviously not a barrier to high-throughput NASBA/TMA as the authors now even state, the Hologic and Biomerieux platforms do seem to work. Perhaps a simple point-of-care or at-home test NASBA would indeed be more practical if the 2-step protocol can be avoided, and the manuscript would be stronger if the authors present that more honestly.

Response:

It is unclear to the authors what changes the reviewer would like us to make, as the

Introduction

“ the 2-step process could be considered inconvenient for high throughput or point of care operation as each additional processing step may present an operational and engineering challenge [14]. “

Discussion

“A truly isothermal, single-step NASBA method is anticipated to enhance NASBA’s potential for adoption in portable diagnostic devices, as it eliminates the need for complex temperature and fluidic control, hence simplifying instrument and operational design. “

and Conclusion

“, removing an operational bottleneck by facilitating one-pot NASBA”

all clearly state the two step process is a bottleneck for decentralised testing, and the aim of this work it so overcome this bottleneck using a SSB.

The pandemic, and particularly Omicron wave has demonstrated that centralised PCR testing is too slow and cumbersome, and rapid antigen testing is adopted despite lower specificity, and higher false positive/false negative rates. Simplicity is the key to effective decentralised testing (see WHO ASSURED criteria), and a single step reaction is simpler than a two-step process. It does not mean the same or superior results cannot be obtained with sophisticated engineering, but if we compare it to the transport sector we have different expectations and are prepared to make different sacrifices when using a plane (complex engineering but centralised) or bicycle (less complex engineering but decentralised) for transport.

Action taken:

To further emphasize the focus of this work towards the field of decentralised, point of care testing, we added

“for enhanced field deployability. “ to line 91

We trust that this explanation combined with the edited text adresses the reviewers concern.

Reviewer #3: Authors of the manuscript entitled “An Improved Nucleic Acid Sequence-Based Amplification Method Mediated by T4 Gene 32 Protein” have thoroughly revised the said manuscript in the light of comments/suggestions raised by the reviewers. Responses to reviewers’ comments have satisfactorily been addressed by the authors and have also been incorporated at the appropriate places within the revised manuscript. The revised manuscript may now be accepted for publication in PLOS ONE. However, there is a couple of very minor corrections that should be done at the Journal level before publication.

Response:

We would like to thank this reviewer for these comments

1. Introduction section on page no. 4, line no. 73- abbreviation ‘RPA’ has been used for the first time in the manuscript, so its full form ‘Recombinase Polymerase Amplification’ should also be written at that place.

Response:

Thanks for pointing out this omission,

Action taken:

We now included the definition before using the abbreviation.

“Compared to other isothermal amplification methods, such as LAMP or Recombinase Polymerase Amplification (RPA), the 2-step process could be considered inconvenient for high throughput or point of care operation as each additional processing step may present an operational and engineering challenge [14].

2. Results and Discussion section, page 5, line 97- ‘times to not include’ should be

‘times do not include’.

Response:

Thanks for pointing out this omission

Action taken:

The typographical error has been corrected and now reads “Please note the recorded amplification times do not include the extra time required for the addition of the amplification enzymes after thermal annealing using the 2-step protocol.”

Submitted filename: Response R2.docx

Click here for additional data file.

22 Feb 2022

24 Feb 2022

We would like to thanks Reviewer 1 for clarifying her/his comments.

Reviewer #1: The authors have again made improvements to the manuscript and adjusted language to be in line with what is actually demonstrated by the data. So I'm mostly in agreement it can be accepted for publication, but there are still a few issues. I said in the last review that claiming the two-step nature of NASBA is the barrier to its widespread use is unfounded, that benefit would be only in an at-home or simple device...and the authors responded with

"It is unclear to the authors what changes the reviewer would like us to make". Well here's one. The very first sentence of the manuscript in the abstract is: "The uptake of Nucleic Acid Sequence-Based Amplification (NASBA) is hindered by the requirement of a thermal denaturation step to initiate the cyclic isothermal amplification." That is plainly not true. If the authors want to cite ASSURED as justification for use of a 1-step vs. 2-step test, they should consider that the E stands for "equipment-free" which doesn't really apply to a test that uses fluorescent beacons no matter how many temperatures are involved.

Response

We would like to thank the reviewer for her/his clarification. and now understand we failed to distinguish between the ultimate goal articulated in the WHO’s Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free and Deliverable to end-users (ASSURED) criteria, and the commonly accepted reality where of point of care diagnostic products often fail to meet part of these criteria, and are not equipment free. We specifically selected reference 1 because within its focus on nucleic acid testing, it articulates the need for

“Procedural simplicity

In order to reduce cost, prevent malfunction and achieve robust, reliable operation within a simple package an amplification technology will preferably be a “single tube” reaction with a minimal volume, employing few reagents and few fluidic manipulations.“ (Craw, LoC, 2012)

The use of gp32 is aimed to simplify the workflow, and we acknowledge this simplification does not eliminate the need for equipment. As we now understand the standpoint of the reviewer, we amended the manuscript to prevent confusion and remove any ambiguity to the field the potential benefits of this work apply to.

Action taken

1. Limited the field the significance of the work applies to point of care diagnostics, and improved the articulation of the scope of the work to the simplification of the work flow by modifying the opening and closing sentence of the abstract

The uptake of Nucleic Acid Sequence-Based Amplification (NASBA) for point of care testing may be hindered by a complexity in the workflow due the requirement of a thermal denaturation step to initiate the cyclic isothermal amplification before the addition of the amplification enzymes.

….

For synthetic HIV-1 RNA, gp32 was found to improve the time-to-positivity (ttp) by average of 13.6% of one-step NASBA and 6.7% of conventional NASBA for the detection of HIV-1 RNA, showing its potential for simplifying the workflow as desirable for point of care applications of NASBA.

2. Clarifying the purpose in line 78 by adding an additional reference to [1]

Procedural simplicity is critical reduce cost, prevent malfunction and achieve robust, reliable operation [1].

3. And re-iterating this in the conclusion

In conclusion, SSBs can be used to decrease the reliance on a thermal annealing step in NASBA, enhancing the procedural simplicity by facilitating one-pot NASBA.

If the authors would simply not claim they've fixed NASBA, a method that's used worldwide every day for diagnostics, but rather keep the claims to that they've shown maybe the denaturation step could be omitted if gp32 is added then I'd be totally fine with this manuscript. The discussion and conclusion paragraph have this much better, so just change the Abstract and I say it's okay.

Response

Now we understand the reviewers misunderstanding of our aim of simplifying the workflow vs meeting the ASSURED criteria including being equipment free, we amended the manuscript as indicated above to clarify the significance of this work is limited to the workflow, and will not allow NASBA to meet he ASSURED criteria. In response to the reviewers wish to amend the abstract, we provide the amended abstract below.

Action taken:

Amended abstract (new text in red)

The uptake of Nucleic Acid Sequence-Based Amplification (NASBA) for point of care testing may be hindered by a complexity in the workflow due the requirement of a thermal denaturation step to initiate the cyclic isothermal amplification before the addition of the amplification enzymes. Despite reports of successful enhancement of other DNA and RNA amplification methods using DNA and RNA binding proteins, this has not been reported for NASBA. Here, three single-stranded binding proteins, RecA, Extreme Thermostable Single-stranded binding protein (ET SSB) and T4 gene gp32 protein (gp32), were incorporated in NASBA protocol and used for single pot, one-step NASBA at 41 °C. Indeed, all SSBs showed significantly improved amplifications compared with the 2-step process, but only gp32 showed no non-specific aberrant amplification, and slightly improved the time-to-positivity in comparison with the conventional NASBA. For synthetic HIV-1 RNA, gp32 was found to improve the time-to-positivity (ttp) by average of 13.6% of one-step NASBA and 6.7% of conventional NASBA for the detection of HIV-1 RNA, showing its potential for simplifying the workflow as desirable for point of care applications of NASBA.

Submitted filename: response v3.docx

Click here for additional data file.

2 Mar 2022

An Improved Nucleic Acid Sequence-Based Amplification Method Mediated by T4 Gene 32 Protein

PONE-D-21-08924R3

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Additional Editor Comments (optional):

After the third round of revision I am pleased that the Authors and the Reviewer have found a point of agreement that allows the acceptance of the manuscript to proceed.

Reviewers' comments:

16 Mar 2022

PONE-D-21-08924R3

An Improved Nucleic Acid Sequence-Based Amplification Method Mediated By T4 Gene 32 Protein

Dear Dr. Guijt:

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