Abstract
Nucleic acid extraction from clinical specimens is essential for accurate molecular diagnostic assays. However, conventional extraction methods are often slow, equipment-dependent, and require trained personnel, limiting the point-of-care applicability. We introduce a novel silica-based pipet tip column that enables rapid nucleic acid extraction from clinical samples in less than 3 min. The pipet tip column has a large surface area for effective nucleic acid extraction using chaotropic salts. The integrated disposable 1 mL syringe eliminates the need for a pipet. Sample application is achieved by simply pushing and pulling the syringe, requiring no external power or specialized equipment. The pipet tip column performance was validated using standard influenza A and B, severe acute respiratory syndrome coronavirus-2, and Staphylococcus aureus samples, achieving high performance even at low concentrations (1 PFU/mL or 1 TCID50/mL). Our approach demonstrated a 90–110% nucleic acid recovery rate, comparable to that of traditional methods. Pipette tip-column nucleic acid extraction was successful for nasopharyngeal and saliva samples, including those in loop-mediated isothermal amplification assays. The pipet tip column provides a rapid, cost-effective, reliable, and portable solution for on-site molecular diagnostics. Additionally, nonexpert users can perform nucleic acid extraction without sample transportation or storage, reducing contamination risks and operational costs.
Introduction
Nucleic acid extraction from clinical specimens is a crucial step in molecular diagnostics, ensuring the accuracy and sensitivity of downstream molecular assays such as polymerase chain and isothermal amplification reactions. Early and accurate diagnosis is vital for controlling infectious agents, such as influenza and SARS-CoV-2, which spread via airborne droplets. , However, clinical samples, such as nasopharyngeal fluids or saliva, often contain inhibitory substances that can reduce diagnostic sensitivity if nucleic acids are not extracted. , Inadequate nucleic acid extraction from clinical samples may degrade target nucleic acids, compromising the reliability of diagnostic results.
Silica-based nucleic acid extraction methods have become a cornerstone in molecular diagnostics owing to their high reliability and efficiency. Silica exhibits a selective binding affinity to the phosphate groups of nucleic acids, especially under chaotropic conditions, which disrupts hydrogen bonding interactions. In the presence of chaotropic salts, such as guanidine and urea, the silica surface becomes negatively charged, facilitating the binding of nucleic acids through cations such as sodium ions. This selective binding mechanism forms the foundation for silica-based resins, which are widely used in commercial nucleic acid extraction kits such as those provided by Qiagen Inc. Silica matrices are employed to adsorb nucleic acids, followed by a series of washing and elution steps, ensuring a high purity and integrity of nucleic acids for molecular diagnostic applications. However, despite their gold standard status for nucleic acid isolation, silica-based extraction methods often require multiple labor-intensive steps, such as centrifugation, pipetting, and washing, which can prolong the process, typically taking up to 1 h, and specialized equipment. − Alternatively, magnetic bead-based extraction offers automation potential and compatibility with high-throughput procedures while being more compact and faster than traditional silica-based methods. However, it still relies on specialized equipment such as magnetic stands, making it less suitable for field applications.
Recent studies have concentrated on the development of equipment-free methods for nucleic acid extraction suitable for on-site diagnostics. The most basic filtration techniques aimed at removing impurities often fail to effectively concentrate nucleic acids, resulting in low yields. These limitations become particularly critical during the early stages of infection, when viral concentrations may fall below detection thresholds. To address these challenges, methods employing chitosan or other substances with binding affinity for nucleic acids are commonly utilized in chromatography techniques using paper or beads, thereby enhancing nucleic acid recovery and concentration. , In addition, recent advancements in microfluidic channels have enabled the integration of sample preparation, nucleic acid extraction, and amplification into a single compact platform, further enhancing the efficiency and speed of on-site diagnostics ,
The accuracy and sensitivity of molecular diagnostics rely on effective nucleic acid extraction from clinical specimens, particularly in the context of controlling infectious diseases such as influenza and SARS-CoV-2. Here, we introduce a novel silica-based pipet tip column designed for rapid and efficient nucleic acid extraction, enabling clinicians and nonexpert users to perform extraction quickly and reliably (Figure ). The objectives of this research were to validate the performance of the tip column against traditional methods and assess its compatibility with standard molecular diagnostic assays, providing a more accessible and effective on-site diagnosis.
1.
Silica-based tip column for on-site extraction of nucleic acids, instead of the conventional centrifugation method. (A) Structure of the silica-based tip column, combined with a disposal syringe by a rubber connector. The column consists of three layers: a glass fiber pad (top), silica beads (middle), and another glass fiber pad (bottom). (B) The manufactured tip column is stored in vacuum packaging after closing the end with a rubber stopper.
Results and Discussion
Optimization of the Buffer Conditions for the Tip Column for Nucleic Acid Extraction
In this study, each buffer for the tip column was optimized by referring to the principles of a commercial nucleic acid purification kit from Qiagen and tested based on previous studies. ,, For the preparation of the lysis buffer, guanidine as a chaotropic salt was saturated as much as possible using ethanol. The amount of viral RNA extracted was higher in buffer with higher concentrations of guanidine than in those with lower concentrations of guanidine, as quantified using RT-qPCR. Finally, 4 M guanidine and 50% ethanol in the lysis buffer were determined to be optimal. In addition, surfactant 1.5% Triton X-100 and 27.5 mM ethylenediaminetetraacetic acid (EDTA) with 12.5 mM Tris-Cl (pH 7.4), used to break virus particles, were included. After extracting RNA from 100 TCID50/mL of SARS-CoV-2 standard samples using the commercial nucleic acid extraction kit from Qiagen and the 1 mL silica-based tip column, we confirmed that the nucleic acid recovery rate with the 1 mL tip column was better. The silica and nucleic acids in the column are bound through charges induced by the chaotropic salt and low concentrations of salts 09t5rrrrrr = 7re required to elute viral RNA in silica. To optimize the elution buffer, NaOH, a base for minimizing the charge interaction between silica and nucleic acids, was tested along with Tris-EDTA and distilled water, which are used as elution buffers in conventional kits with low salt concentrations. As a result, we confirmed the optimized concentration of NaOH as the elution buffer. The 2 mM NaOH used in the subsequent experiment was within the concentration range and, thus, did not affect the PCR (Figure S2).
Extraction of Genomic Nucleic Acids from Influenza A and B, SARS-CoV-2, and Staphylococcus aureus Standards
Each sample was prepared by serially diluting the influenza A and B standard particles and heat-inactivated SARS-CoV-2 standard particles purchased from ATCC at 0, 1, 10, and 100 TCID50/mL using a viral transport medium (VTM). Each RNA sample was extracted using the Qiagen nucleic acid extraction kit as a comparative group and a 1 mL tip column and diluted with 250 μL of VTM. The cycle quantification (Cq) values were calculated using RT-qPCR to confirm nucleic acid recovery. The genomic DNA of S. aureus standards from ATCC diluted with PBS was also extracted and detected using qPCR (Figure ). The Cq values were 1–3 Cq higher than those of samples obtained using the Qiagen kit, with 90–100% recovery for each nucleic acid, but the yield was higher in samples with low concentrations requiring high sensitivity (Figure S3). It is expected that the nucleic acid extraction efficiency is related to the density of the silica-based column, and unlike conventional methods that utilize high centrifugal force, it showed a lower nucleic acid recovery rate due to the condition that the solution had to be filtered only by the pressure of the syringe. Furthermore, compared with the commercial kit, which requires approximately 40 min of pipetting and centrifugation, the tip column is more advantageous for point-of-care molecular diagnosis because it takes approximately 3 min and can be used once without any electronic machines.
2.
Comparison of Cq values of nucleic acid extraction recovery from influenza A and B, SARS-CoV-2, and S. aureus standard samples between the Qiagen nucleic acid extraction kit (black) and the tip column (red). Each standard sample concentration on the X-axis represents the concentration of the initial sample before the lysis step. The results represent the average and standard deviation calculated from triplicate. Cq, cycle quantification.
Quantification of Extracted Nucleic Acids Using qPCR and Detection
The extraction of the same 15 standard samples using both the Qiagen kit and the tip column showed no significant differences in performance. The coefficient of determination (R 2) was calculated to be 0.9728, indicating a very high correlation between the two methods and demonstrating their comparable efficiency in nucleic acid extraction from standard samples (Figure A). The clinical samples collected from the swab were stored in 2–3 mL of VTM at a concentration ∼50 times lower than that of the initial fluid. Nevertheless, for the clinical samples that were not subjected to nucleic acid extraction, the presence of substances such as mucin, RNase, and other inhibitors that interfere with PCR led to many positive samples exhibiting high Cq values or not detected (Cq > 40). In contrast, 13 positive clinical samples were successfully identified as positive using both the Qiagen kit and the tip column (Figure B). In the case of clinical samples, the correlation was lower than that of standard samples, with a R 2 value of 0.8439, reflecting the challenges posed by sample complexity and impurities in the clinical matrix.
3.
Correlation of viral RNA extraction efficiency between the Qiagen kit and tip column in (A) 15 standard samples of influenza A and B and SARS-CoV-2 and (B) 13 clinical samples of influenza A. Nine of the 13 clinical samples were not sufficiently amplified in 40 cycles of RT-qPCR (gray dotted line, not detected). The arrows indicate differences in trend lines between nucleic acid extraction by the Qiagen commercial kit and the tip column in standard or clinical samples.
To enhance diagnostic accuracy, it is crucial to recover higher concentrations of target nucleic acids. Previously, the method involved resuspending the swab in VTM before extracting viral RNA, enabling the use of clinical samples with relatively diluted concentrations. In this study, viral RNA was extracted directly by immersing the entire undiluted saliva swab containing the virus in lysis buffer. The presence of saliva (even 1 μL), which contains the target nucleic acids, can interfere with PCR, rendering detection impossible (Figure S4). For viral RNA extraction, saliva samples with 10 TCID50/mL of SARS-CoV-2 were added to 1 mL of lysis buffer (Figure S5). The lowest Cq value was obtained when applying a 50 μL salivary sample; applying ≥100 μL of the sample increased the syringe pressure, preventing efficient lysate collection. Additionally, most commercial nasal swabs were sufficiently saturated with approximately 50 μL. To verify the versatility of the tip column for viral RNA and other nucleic acid extraction, genomic DNA from S. aureus was extracted after dilution in PBS. The extracted DNA was quantified using qPCR and compared with DNA extracted using a commercial Qiagen kit, and comparable results were obtained. Therefore, the tip column proved to be effective for extracting different types of nucleic acids across various targets.
To assess the reproducibility and stability of the tip columns, we manufactured them in a single batch and conducted repeated tests. The 20 tip columns produced on the same day exhibited high reproducibility, with a coefficient of variation of <1.83%. Accelerated stability testing at 37 °C indicated consistent performance, with a coefficient of variation ≤1.72%, maintaining stability for up to 1 year at room temperature (25 °C) (Figure ).
4.
Maintenance and reproducibility of the tip column. The performance of nucleic acid extraction was maintained for 1 year at room temperature (25 °C) at approximately 500 TCID50/mL SARS-CoV-2 (CV: 1.76%), and a similar Cq value was derived each time on the day of tip column production (CV: 1.83%). CV; coefficient of variation. AVG; average. STD; standard deviation.
Loop-Mediated Isothermal Amplification (LAMP) Detection of SARS-CoV-2 RNA Extracted Using the Tip Column
To apply the tip column as a point-of-care test instead of the RT-qPCR available at the lab level, we compared the tip column with the commercial SARS-CoV-2 LAMP kit from mMonitor. The sensitivity of the kit is 25 copies/reaction or 16.7 TCID50/mL. The LAMP kit requires the use of purified SARS-CoV-2 RNA as a template. After the SARS-CoV-2 RNA was extracted using the commercial nucleic acid extraction kit from Qiagen as a positive control and a 1 mL tip column in this study, it was observed that 100 TCID50/mL of the SARS-CoV-2 sample could be distinguished from the negative control by the colorimetric signals (Figure ). The test reliably detected viral RNA even at low concentrations, and the results were visually clear, with positive samples changing color from clear to blue. This color change directly correlates with the presence of the target nucleic acid, providing a straightforward and rapid visual confirmation of the presence of SARS-CoV-2. The LAMP kit changes color from clear to blue in positive samples, indicating the presence of the target nucleic acid. The technique is compatible with the conventional PCR and isothermal amplification methods, such as LAMP and recombinase polymerase amplification, significantly enhancing the robustness and reproducibility of these assays by mitigating the inhibitory effects of sample impurities and ensuring consistent performance even in the presence of complex biological samples.
5.
LAMP analysis results using the MmaxSure IVD REF EZ SARS-CoV-2 detection kit with 100 TCID50/mL of SARS-CoV-2. Each viral RNA as a template was extracted using the Qiagen RNA extraction kit or the tip column from SARS-CoV-2 spiked into saliva. The intercalating colorimetric dye contained in the LAMP premix changes from colorless to blue in positive samples.
Conclusions
Rapid and user-friendly nucleic acid extraction methods are essential for molecular diagnosis, even from a long-term perspective. There have been attempts to detect viruses through molecular diagnostics without a nucleic acid extraction step for point-of-care testing. The tip column was developed to extract nucleic acids directly from clinical samples in the field of molecular diagnosis. It can be easily assembled by connecting a silica-based column to a disposable syringe with a rubber connector. Unlike traditional extraction kits that require centrifugation, nucleic acids can be purified by applying the lysate, wash buffer, and elution buffer with a disposable syringe connected to the tip column. The extraction kit developed in this study offers a simple, single-use process that minimizes the risk of contamination and is user-friendly, even for nonspecialists. The need for portable nucleic acid extraction devices has become increasingly important for on-site applications, especially in resource-limited environments where accurate diagnostic approaches are crucial. , The Biomeme M1 Sample Prep Kit, designed for molecular diagnostics, has an elution volume of 0.85 mL, which may lead to dilution of nucleic acids and reduced analytical sensitivity. , Additionally, the extraction process is labor-intensive, requiring seven steps and more than 39 pump actions, and lacks data on recovery rates, raising concerns about its efficacy in certain applications. − In contrast, the tip column we developed significantly lowers the elution volume to 60 μL, increasing the nucleic acid concentration and improving target analyte recovery. This advancement makes our system more suitable for applications requiring high sensitivity, effectively addressing the growing demand for efficient nucleic acid extraction methods in field diagnostics.
The extracted nucleic acids can be quantified using molecular diagnostic methods, such as qPCR. We confirmed that the tip column is highly comparable to commercial nucleic acid purification kits for influenza A and B, SARS-CoV-2, and S. aureus in terms of recovery. Thus, the silica-based tip column provides an easy method for extracting RNA or DNA from clinical samples and is expected to be more advantageous for on-site molecular diagnostics than centrifugation-based methods, particularly for nasopharyngeal and saliva samples.
Methods
Chemicals and Instruments
Silica (75–90 nm), guanidine thiocyanate, sodium hydroxide, EDTA, antimouse immunoglobulin G antibody, and skim milk powder were purchased from Sigma-Aldrich (St. Louis, MO, USA). The disposable pipet tip was purchased from PakGent Bioscience (Suzhou, Jiangsu, China). Ethanol, Triton X-100, 1 M Tris-Cl buffer (pH 7.4), QIAmp Viral RNA Extraction Kit, and QIAamp DNA Mini Kit were purchased from Qiagen Inc. (Hilden, Germany). The TOPreal One-step RT qPCR Kit (TaqMan Probe) and TOPreal qPCR 2X Premix (TaqMan Probe) were purchased from Enzynomics (Daejeon, South Korea). Standard influenza A and B virus particles were purchased from the Korea Bank for Pathogenic Virus (Seoul, South Korea). Clinical nasopharyngeal samples of influenza A and B were purchased from Discovery Life Sciences (Huntsville, AL, USA). SARS-CoV-2 culture fluid (heat-inactivated) was purchased from Zeptomatrix (Buffalo, NY, USA). Oligonucleotide primers were synthesized by Zenotech Corp. (Daejeon, South Korea). − Clinical samples were purchased from Discovery Life Sciences. The SARS-CoV-2 LAMP Kit was purchased from Mmonitor (Daegu, South Korea).
Design of a Silica-Based Tip Column for Nucleic Acid Extraction
A disposable pipet tip column was developed for nucleic acid extraction, containing silica beads and silica-based glass fiber pads arranged to facilitate efficient binding and purification. The silica beads and glass fiber pads were strategically placed within the pipet tip to maximize nucleic acid recovery from clinical samples under chaotropic conditions.
To enhance the ease of use in field applications, the tip column was integrated with a disposable syringe via a rubber connector, eliminating the need for external power or complex laboratory equipment. This design offers a user-friendly, cost-effective, and disposable solution for rapid nucleic acid extraction directly from clinical specimens.
Preparation of Silica Beads
Silica beads (75–90 mm) were treated with a piranha solution for the activation of silanol groups and washed with distilled water over 20 times. A layer of silica beads creates a higher density and resistance at the binding site, which induces better nucleic acid binding to the column, than those with glass pads. Inside the 1 mL disposable tip, 45 μL of silanol group-activated silica beads was placed on the bottom glass pad, removing as much moisture as possible. The size of the glass pads was 4 × 5 mm for the bottom of the silica beads and 5 × 10 mm for the top of the silica beads. The tip column combination was stored in vacuum packaging (Figure b). Before nucleic acid extraction, lysis buffer (4 M guanidine thiocyanate), 1.5% Triton X-100, 50% ethanol, and 12.5 mM EDTA in 27.5 mM Tris-Cl (pH 7.4), wash buffer [50% ethanol in 1 mM Tris-Cl (pH 7.4)], and elution buffer (2 mM NaOH) were prepared.
Tip-Column Extraction of Nucleic Acids Using Standard and Clinical Samples
The nucleic acid extraction process using the silica-based tip column involves a streamlined four-step protocol as illustrated in Figure , lysis, binding, washing, and elution. Initially, clinical nasopharyngeal samples are combined with the lysis buffer containing chaotropic salts to denature proteins and disrupt cellular structures. The lysis buffer comprises a high concentration of guanidine thiocyanate, a chaotropic agent that facilitates efficient nucleic acid binding to silica by disrupting hydrogen bonding. The presence of ethanol, with its higher dielectric constant than that of water, dehydrates nucleic acids by removing surrounding water molecules, allowing positively charged ions to interact with nucleic acids and form salts. This interaction is critical; in the absence of ethanol or isopropanol, the phosphate groups of nucleic acids remain encased in water, impeding access to these ions.
6.
Procedure for nucleic acid extraction in 3 min using the silica-based tip column combined with a disposable syringe. The extraction process consists of pulling up and pushing down the lysate and binding, washing, and eluting buffers sequentially into the tip column via the connected syringe. The purified nucleic acids could be subjected to molecular diagnosis as templates.
Following the lysis step, an appropriate volume of lysate and buffers is drawn into the tip column by using a disposable syringe. The optimized sample-to-lysis buffer ratio, determined through preliminary experiments, involves mixing 250 μL of either the virus standard or clinical sample with 850 μL of lysis buffer (Figure S1). This mixture is then allowed to interact with the silica-based matrix within the tip column. Any unbound impurities are efficiently removed by applying a positive pressure to the syringe, thereby eliminating the necessity for centrifugation, which is often impractical in on-site extraction scenarios.
In the subsequent washing phase, 1 mL of a wash solution comprising 50% ethanol is sequentially drawn up and down through the tip column. This step is crucial for maintaining the binding interactions between nucleic acids and silica while effectively washing away nonspecifically bound contaminants. However, it is important to acknowledge that residual alcohol can adversely affect subsequent polymerase chain reaction (PCR) amplification steps. After washing, we use a syringe to apply air pressure as a pump three to four times to remove as much residual alcohol as possible between the washing and elution steps, ensuring effective removal of alcohol in the silica layer. Most conventional DNA and RNA extraction kits address this limitation by employing centrifugation to ensure complete removal of alcohol; however, this method is not feasible for on-site applications.
In the final elution step, 60 μL of an elution buffer is introduced into the tip column to disrupt the binding interactions between nucleic acids and silica, facilitating the release of the bound nucleic acids. The eluted nucleic acids are then collected in a new tube, allowing for their storage and utilization in subsequent diagnostic assays. The entire extraction process is designed to be completed within 3 min without the need for specialized machinery or external power sources, thereby enhancing the feasibility of rapid molecular diagnostics in diverse clinical settings.
We obtained influenza A, influenza B, and SARS-CoV-2 virus-infected patient samples from Discovery Life Sciences. This study was approved by the Institutional Review Board (IRB) of the Gwangju Institute of Science and Technology, which determined that the study was exempt from further IRB review. The IRB concluded that the research posed no issues related to the protection of personal data, adherence to bioethics, or safety, as the collection of saliva samples was noninvasive and carried minimal risk. Additionally, the study was recognized to have sufficient academic and ethical justification. The biofluid samples were stored at – 80 °C for subsequent analysis. We used patient nasal fluid samples to confirm the recovery rate of the nucleic acid extraction methods according to the procedure described above.
Quantification of the Extracted Nucleic Acids Using Quantitative PCR (qPCR) and Detection with LAMP
The extraction efficiency was compared by quantifying the nucleic acid extracted from the standard sample by using the RT-qPCR method. The primer sequences for quantitative analysis for the detection of influenza A and B, SARS-CoV-2, and S. aureus are listed in Table . ,− RNA from influenza A and B and SARS-CoV-2, and genomic DNA from S. aureus standards, were extracted using the QIAamp DNA Mini Kit for comparison with the tip column method. Thereafter, 4 μL of the extracted viral RNA was added to an RT-qPCR mixture comprising 1 μM primer and 1× TOPreal One-step RT-qPCR reaction mix (TaqMan probe), with a final reaction volume of 20 μL. RT-qPCR under optimal conditions was performed on the CFX Connect Real-Time System as follows: reverse transcription for 30 min at 50 °C, initial denaturation for 10 min at 95 °C, followed by 40 elongation/extension cycles at 95 °C for 15 s and 60 °C for 60 s.
1. Primer Sets for the Quantification of Nucleic Acids Extracted from Influenza A and B and Genomic RNA from SARS-CoV-2 Using RT-qPCR, as Well as Genomic DNA Extracted from Staphylococcus aureus Using qPCR.
| Target | designation | sequence (5′–3′) | ref |
|---|---|---|---|
| influenza A (Mgene) | forward | CTT CTA ACC GAG GTC GAA ACG TA | |
| reverse | GGT GAC AGG ATT GGT CTT GTC TTT A | ||
| probe | FAMTCA GGC CCC CTC AAA GCC GAGBHQ1 | ||
| influenza B (HA gene) | forward | AAA TAC GGT GGA TTA AAC AAA AGC AA | |
| reverse | CCA GCA ATA GCT CCG AAG AAA | ||
| probe | FAMCAC CCA TAT TGG GCA ATT TCC TAT GGCBHQ1 | ||
| SARS-CoV-2 (N gene) | forward | TTA CAA ACA TTG GCC GCA AA | |
| reverse | GCG CGA CAT TCC GAA | ||
| probe | FAMACA ATT TGC CCC CAG CGC TTC AGBHQ1 | ||
| Staphylococcus aureus (femA gene) | forward | ACT GTG ACG ATG AAT GCG ACA A | |
| reverse | ATG TTG TGG TGT TCT TAT ACC AAA TCC | ||
| probe | FAMCGA CAA CTG GCA CAT TGG CTA TCG CTT TBHQ1 |
Subsequently, 4 μL of the extracted genomic DNA from S. aureus was detected using qPCR with a mixture comprising 1 μM primer and TOPreal qPCR 2× Premix (TaqMan Probe), with a final reaction volume of 20 μL. The recommended qPCR conditions were used as follows: initial denaturation for 10 min at 95 °C and then 40 elongation/extension cycles at 95 °C for 15 s and 60 °C for 60 s. The fluorescence data were collected in the FAM channel during each elongation/extension cycle. All reactions were performed in triplicate. The addition of NaOH did not affect the sensitivity of the reaction. For on-site molecular diagnosis, 4 μL of the extracted nucleic acids eluted with 2 mM NaOH was used as a template with LAMP for SARS-CoV-2 detection. To evaluate the applicability of on-site molecular diagnostic kits, we used a commercially available SARS-CoV-2 LAMP kit from Mmonitor. The LAMP reaction was performed for 40 min at 60 °C, and a change from colorless to blue was observed.
Supplementary Material
Acknowledgments
This work was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea [grant number: HW20C2057]; National R&D Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (2021M3C1C-3097694); and Materials and Components Technology Development R&D project (RS-2024-00508627) funded by the Ministry of Trade Industry & Energy (MOTIE, South Korea).
Glossary
Abbreviations
- Cq
cycle quantification
- EDTA
ethylenediaminetetraacetic acid
- IRB
Institutional Review Board
- LAMP
loop-mediated isothermal amplification
- VTM
viral transport medium
All data generated or analyzed during this study are included in this published article (and its Supporting Information files).
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c00573.
Optimization of the ratios of sample/lysis buffer with the same volume of lysate for nucleic acid extraction applied by the tip column; optimization of the elution buffer with 100 PFU/mL of influenza A; comparison of Cq values for nucleic acid extraction recovery of Influenza A, B, SARS-CoV-2, and S. aureus standard samples between the Qiagen kit and the tip column; effect of saliva volume on RT-qPCR with 500 TCID50/mL SARS-CoV-2 without nucleic acid extraction; and RT-qPCR results for clinical salivary sample volume optimization tests with 850 μL of lysis buffer for the tip column extraction (PDF)
§.
Division of emerging infectious disease, Korea Disease Control and Prevention Agency (KDCA), 187 Osongsaengmyeong 2-ro, Cheongju-si, Chungcheongbuk-do, 28159, Republic of Korea
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Chaewon Jung: writingoriginal draft. Experiments. Troubleshooting. Gyeohoon Kim: Investigation (sample preparation and clinical sample testing). Mun-Beom Song: methodology (tip column creation). Min-Gon Kim: Supervision. Research support. Manuscript review.
The authors declare no competing financial interest.
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Supplementary Materials
Data Availability Statement
All data generated or analyzed during this study are included in this published article (and its Supporting Information files).