The Baculovirus Expression Vector System (BEVS) is extensively used for expressing target proteins, necessitating accurate baculovirus titration to ensure the output. Traditional plaque assay assisted titration, while reliable, is labor-intensive and time consuming. Here, we introduce an improved quantitative real-time polymerase chain reaction (qPCR)-based method for determining baculovirus titers. Briefly, target-gene specific primers are designed to produce a 180-bp PCR product using viral DNA as the template. The PCR products are purified and the mass weight of the PCR products are quantified based on 260-nm absorbance. Based on the molecular weight of the 180bp PCR product, the mass weight is then converted to the molar concentration which is finally converted to molecule number by applying the Avogadro constant. Such PCR product is serially diluted and used as the template for qPCR. The qPCR Ct values between serial dilutions are used to generate the standard curve. Finally, the Avogadro’s number (i.e. titer) of the test viral DNA is inferred from its qPCR Ct value by comparing with the standard curve. Our method eliminates the need for a pre-titrated viral stock, reducing variability caused by storage conditions. This advancement is particularly beneficial for large-scale production of the target protein using the BEVS.
Article
A rapid and accurate method for determining titers of baculovirus for protein expression
https://doi.org/10.21203/rs.3.rs-5000511/v1
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Avogadro’s number
Baculovirus Expression Vector System (BEVS)
baculovirus titration
bac-to-bac
quantitative Real-Time PCR (qPCR)
SYBR Green
The Baculovirus Expression Vector System (BEVS) is an effective platform for expressing large-scale of target proteins1, especially for generating high yields of eukaryotic proteins with characteristics that closely mimic those found in nature2. When using this system, accurate baculovirus titration is essential for achieving optimal protein yields. Traditionally, plaque assay has served as the authentic approach for titration3, but its labor-intensive and time-consuming nature has impeded the progress of research. To overcome these limitations, alternative methods, such as quantitative real-time polymerase chain reaction (qPCR), have been developed. The principle of qPCR4 is based on the use of fluorogenic TaqMan probes5 or dyes like SYBR Green6, which measure the intensity of a fluorescent signal corresponding to the amount of DNA yielded during PCR amplification7. SYBR Green is particularly useful as it emits a bright fluorescence upon specifically bound to double-stranded DNA (dsDNA), and the fluorescence intensity increases proportionally with the amount of amplicon. The qPCR-based methods offer a more efficient and convenient approach to baculovirus titration, thereby promoting the application of BEVS8–10. However, since the qPCR-based methods could only give a relative concentration of the template in the reaction, the previously reported qPCR-based method requires the known titer of the viral stock for generating the standard curve. The titer of the test virus is then estimated by pinpointing the Ct value of test viral DNA on the standard curve8;9. The bottleneck is that, in most of the time, the titer of a viral stock is unknown. More importantly, the titer of a viral stock is changing with time during storage, thus severely interfere with the accurate titration.
We report here an improved qPCR-based method for determining baculovirus titers. Unlike previously reported qPCR-based titrating methods8;9, our approach does not require known titers of the viral stocks for generating the standard curve. In brief, first, gene specific primers were designed for obtaining PCR products using the viral DNA as a template. This PCR product was a 180-bp fragment that corresponds to the gene sequence of interest, ensuring the titration of the desired virus rather than the Defective Interfering Particles (DIPs)11;12, which often lack the inserted gene of interest. The PCR products were quantified for obtaining the molar concentration (thus the Avogadro’s number) and were then subjected to 10x serial dilution. The serially diluted PCR products with known Avogadro’s number were used as the template for the qPCR using the same primers and the obtained Ct values were used for constructing the standard curve. Meanwhile, the same gene-specific primers were utilized to amplify the DNA fragment from the test virus stock, and the concentration/titer of the viral DNA sample is inferred from its Ct value based on the standard curve. Our improved qPCR-based baculovirus titration method allows for rapid and accurate determination of baculovirus titers, without the need for cell seeding on plates or 10x dilutions of virus stocks prior to infection. This method has been validated as highly effective for baculovirus titer determination through numerous times of large-scale protein production practice in our work.
Generating standard curve using 10x serially diluted DNA with known concentration
The major steps involved in our method are outlined and paralleled with the previously reported methods8;9 in Fig. 1.
The first step of our method was to design gene specific primers to amplify a 180-bp DNA fragment of known sequence using the plasmid or viral DNA harboring the target gene as the template. In this report, we adopted the BEVS to express two human genes, namely Digestive-organ Expansion Factor (DEF) and a mutant form of CALPAIN3 called CAPN3C129S for the purpose to unravel the structural basis of the interaction between DEF and CAPN3 proteins13–16. Following purification, the mass weight concentration of the PCR product was measured using a NanoDrop apparatus, and the molar concentration of the PCR products was calculated based on the mass weight concentration versus the molecular weight of the 180-bp DNA fragment. The Avogadro’s constant was then applied to obtain the molecular number of the PCR product in one milliliter (mL). Such PCR product of known concentration was subjected to 10x serial dilutions (now with known Avogadro’s number for each dilution) and the dilutes were used as the template for new qPCR using the same primers. The Ct values for each dilute were used to construct the standard curve (Fig. 1). In conjunction with amplification of the same 180-bp DNA fragment using the test viral DNA as the template, we can determine the actual baculovirus titer simply by comparing the Ct value of the test sample with the standard curve (Fig. 1).
When performing the qPCR reactions, due to the saturated binding of the SYBR Green dye by the excessive amounts of DNA templates in the solutions of initial PCR product and first two dilutes, it was not surprising to find that the qPCR using these three templates often failed to yield a Ct value, manifested as the result “N/A” by the qPCR machine (Table 1). The qPCR of the remaining dilutes not only yielded a reproducible Ct value in three replicates for each dilute but the increase of the Ct values showed a nice correlation with the factor of 10x serial dilution as well (Table 1).
| Group | Well | Ct | average Ct |
|---|---|---|---|
| cont-0 | A01 | N/A | |
| cont-0 | A02 | N/A | N/A |
| cont-0 | A03 | N/A | |
| cont-1 | A04 | N/A | |
| cont-1 | A05 | N/A | N/A |
| cont-1 | A06 | N/A | |
| cont-2 | A07 | N/A | |
| cont-2 | A08 | N/A | N/A |
| cont-2 | A09 | N/A | |
| cont-3 | A10 | 4.48 | 4.86 |
| cont-3 | A11 | 4.95 | |
| cont-3 | A12 | 5.15 | |
| cont-4 | B01 | 10.06 | 10.22 |
| cont-4 | B02 | 10.36 | |
| cont-4 | B03 | 10.24 | |
| cont-5 | B04 | 14.4 | 13.82 |
| cont-5 | B05 | 13.52 | |
| cont-5 | B06 | 13.53 | |
| cont-6 | B07 | 17.27 | 17.20 |
| cont-6 | B08 | 17.21 | |
| cont-6 | B09 | 17.13 | |
| CD-S1 | B10 | 21.04 | 21.06 |
| CD-S1 | B11 | 21.01 | |
| CD-S1 | B12 | 21.14 | |
| His.Def-S1 | C01 | 18.42 | 22.27 |
| His.Def-S1 | C02 | 30.23 | |
| His.Def-S1 | C03 | 18.15 | |
| His.Def-S2 | C04 | 21.83 | 21.51 |
| His.Def-S2 | C05 | 21.21 | |
| His.Def-S2 | C06 | 21.48 | |
| CD-S2 | C07 | 16.17 | 16.22 |
| CD-S2 | C08 | 16.1 | |
| CD-S2 | C09 | 16.38 | |
| Note: Group cont-0 represents the concentration-known original control PCR product without any dilution. Group cont-1 represents a 10x dilution of cont-0, group cont-2 represents a 10x dilution of cont-1, and so on. The groups of CD-S1 and CD-S2 represent two different batches of the CD virus, while groups of His.Def-S1 and His.Def-S2 represent two different batches of the His.Def virus. Both CD virus and His.Def virus contain the target gene DEF (HGNC: 28440; NCBI Gene: 27042; Ensembl: ENSG00000117597). Here, the cont-0 PCR product was obtained using the CD donor plasmid as a template. The viral DNA was used as the template for obtaining the control PCR products in our later experiments. | |||
Illustrated as below (Fig. 2), utilizing the Ct value of each 10x serial dilute of the gene specific PCR products with known concentration to generate standard curve, we can now determine the actual baculovirus titers without knowing the initial titer of the viral stock.
As shown in Fig. 2, consistent with the theoretic prediction, the standard curve displayed a nice linear relationship between the Ct values and log values of the molecular number serving as the template in the qPCR process. The line equation was accordingly generated. Therefore, we could now directly substitute the Ct value of the test viral DNA into this equation to calculate the corresponding log value to obtain the molecule number of the test viral DNA in 1mL, namely the titer of the baculovirus. Following is the example in our practice (Table 2).
| viral ID | Ct value | log value | Molecule number in 5 µL viral stock | Titer (molecule number in 1mL viral stock) |
|---|---|---|---|---|
| His.Def-S1 | 22.26 | 4.614 | 4.12x104 | 8.23x106 |
| CD-S2 | 16.21 | 6.084 | 1.21x106 | 2.43x108 |
| His.Def-S2 | 21.50 | 4.799 | 6.30x104 | 1.26x107 |
| CD-S1 | 21.06 | 4.907 | 8.07x104 | 1.61x107 |
| Note: Viral ID is used to differentiate different test viral stocks containing the DEF target gene. The Ct value of each test viral DNA subjected to the qPCR analysis was plotted against the standard curve to obtain the corresponding molecule number which is shown in the “molecule number in 5µL” column. The titer of the test virus is then calculated and shown as the molecule number in 1mL. | ||||
Application of the improved qPCR-based method reveals the titer decrease caused by the viral stock storage
It would be ideal to compare our titration method with the authentic plaque assay. However, considering that our method was simply improved from previously reported qPCR-based methods which have been vigorously validated against plaque assay8; 9, we thought it was not a requirement to perform such a comparison in our case. In fact, we were more eager to learn whether our method could be applied to detect the changes of viral titers caused by storage, a factor often related to the reduced protein yield in practice. To this end, we titrated four viral stocks at two different time points of storage.
| Viral ID | 1st titration | 2nd titration | Titer Decreased | ||
|---|---|---|---|---|---|
| date | titer | date | titer | ||
| CD-S3(a) | 2023.05.30 | 3.46×109 | 2023.06.13 | 8.50×108 | 75.4% |
| CD-S4(b) | 2023.03.02 | 5.97×109 | 2023.04.29 | 1.50×108 | 97.5% |
| R3-S1(c) | 2023.11.06 | 1.03×107 | 2023.12.26 | 1.68×106 | 83.7% |
| R3-S2(d) | 2024.05.06 | 3.62×109 | 2024.07.22 | 2.60×109 | 28.2% |
| Note: (a) and (b) are two different batches of the CD viral stocks, and (c) and (d) are two different batches of the R3 viral stocks. R3 is the virus derived from recombinant donor plasmid of pFastBac HT C containing the His.DEF gene and CAPN3C129S gene17, fused together to form an Open Reading Frame. Titer Decrease is defined as (1st titrated titer – 2nd titrated titer) ÷ (1st titrated titer) × 100%. | |||||
As seen in Table 3, the titers of all four viral stocks decreased obviously after certain period of storage. We have shown in the above that our method ensures the qPCR efficiency to be relatively constant for a given 180-bp PCR product, considering the titer of a viral stock tended to decline with time of storage, and the time interval between the two measurements exceeded 13 days, we thought the dramatic decrease in titers was a naturally-occurring process, instead of the inaccuracy of this method. The observed decreasing viral stock titers with time contrasted to the previously reported qPCR-based method in which the naturally-occurring titer decline could not be detected9. According to the official manual provided by Thermo Scientific and all the preceding practices in our laboratory and other researchers, it is almost certain that the baculovirus titer would decrease during storage. The inability to detect the titer drop during storage can render the use of baculoviruses stored longer than a few months highly unreliable. During our practice, we noticed that, after certain period of storage, the viral stock produced visible precipitates at the bottom of the tube (Fig. S1), which might explain the decreasing titers during storage, however, the exact reason remains elusive to us. Initially, we thought the precipitates were resulted from the bacterial contamination, but this speculation was proved not to be the case since the harvest of the cells infected with the viral stock containing the noticeable precipitates did not contain any bacteria (data not shown). Interestingly, the titer of the R3-S2(d) viral stock did not show a pronounced decrease compared to other three groups. This was probably attributed to the addition of 10% Fetal Bovine Serum (FBS) to the R3-S2(d) viral stock but not to other three viral stocks to protect the virus against proteinases in the medium. However, the exact reason remains unknown.
Suggested virus dosage for large-scale protein production based on viral titer
Multiplicity of Infection (MOI) is commonly used to guide the usage of virus for infection. MOI is the ratio of the number of viral particles (viral titer times viral volume) to the number of host cells, and the calculation formula is:
Here, titer refers to the viral titer calculated earlier; vv denotes the volume of viral stock added; cd represents cell density calculated using a hemocytometer (or other counting device); cv stands for cell culture volume. According to our experience, when expressing proteins on a large scale, baculovirus-based expressions of different target proteins have varying optimal MOIs, but generally around 10. We therefore recommend to determine the optimal MOI to express the protein of interest by testing at least 5, 10, 15 three MOIs. Subsequent experiments can then be conducted using the optimal MOI determined. For example, for a 300mL culture system, if the cell density is 2.5×106 cells/mL and the viral titer is 5×108 pfu/mL, setting the MOI equal to 10 yields:
Therefore, the required volume of viral stock is 15mL.
Prior to the development of this improved qPCR-based titration method, due to the inconvenience of plague assay and the control group variability of the previously reported qPCR-based methods, we arbitrarily added 10 mL of the viral stock into 300 mL cells with a cell density of 3.0 x 106. The expression of target protein often resulted in quite low yields, significantly impeding our research progress. In contrast, application of our improved qPCR-based titration method has consistently achieved optimal expression effects. In the following, we provided three examples of protein expression experiments using the virus titrated with our improved qPCR-based method (Table 4 and Fig. 3). In these cases, the volume of the virus stock we added was calculated according to the above equation. Two days post the virus infection, we harvested the Sf9 cells in suspension and performed ultrasonic treatment and centrifugation to obtain the supernatant containing the expressed protein. The supernatant was subjected to the affinity-based purification using Ni-NTA agarose beads. Coomassie blue staining showed that the target protein CAPN3C129S and DEF were successfully expressed by the Sf9 cells transfected with the His.C129S-S1 and His.Def-S3 virus, respectively (Fig. 3A,B). The CD virus could co-express His.DEF and CAPN3C129S two target proteins. Because DEF interacts with CAPN3, the CAPN3C129S protein was co-purified when using the Ni-NTA beads to purify the His.DEF protein, and that explains the two proteins bands observed in the purified products. It was evident that the expression of the target protein was satisfactory in all cases, demonstrating the reliability of our titrating method.
| Viral ID | Titer | MOI | Expression Result |
|---|---|---|---|
| His.C129S-S1 | 1.24×109 | 13.2 | Satisfactory |
| CD-S5 | 1.11×109 | 10.0 | Satisfactory |
| His.Def-S3 | 1.10×109 | 9.0 | Satisfactory |
We report here an improved qPCR-based method for baculovirus titration and have demonstrated the efficiency and reliability of this method. However, how to improve this method further remain to be explored. For example, we attempted to further simplify this method by omitting the viral DNA extraction step, but failed in producing the PCR products. Considering that the viral particles are encapsuled in the proteinaceous shell, susceptible to protease digestion, we also tried to treat baculovirus with protease K but the product was ineffective to serve as a template in PCR amplification (data not shown).
SYBR Green dye-based method is widely used due to its simplicity and low cost. However, its major drawback lies in its lack of specificity, as the dye can bind to any dsDNA. Therefore, non-specific amplification products in qPCR can increase fluorescence values, affecting the accuracy of quantitative results. To address this problem, the TaqMan-probe based method can be applied to mitigate the non-specificity issue. Nevertheless, our method still employs the SYBR Green dye-based method because we reckoned that the non-specificity issue of SYBR Green only significantly affects results when the relative amount of dsDNA to be quantified is low, which implies a low titer of the virus stock under examination. In practice, if the virus titer is too low, it is considered unusable and thus such stock is normally discarded. Only when the virus titer reaches more than ~ 1x107 can the viral stock deem to be adequate for use, and within this range, quantification of qPCR product using SYBR Green is reasonably accurate.
Primer sequence
In this paper, we adopted the BEVS to express DEF and CAPN3C129S two target proteins13–16 in Sf9 cells, therefore, primer pairs specific to these two genes were designed to amplify corresponding 180-bp DNA fragments from the virus inserted with these two genes, respectively (Table S1). Fig. S2 shows an example of constructing the donor plasmid of the His.Def virus.
Generation of bacmid by using the Bac-to-Bac system
The principle and workflow for using the Bac-to-Bac system to express target proteins is illustrated in Bac-to-Bac® Baculovirus Expression System (available from: www.invitrogen.com). In brief, after the gene of interest was cloned into the pFastBac plasmid, the recombinant plasmid was transformed into DH10Bac E. coli cells containing the bacmid, which allow the transposition of the gene of interest into the bacmid. After the antibiotic selection, the positive colony was obtained and used for further cultivation. The bacmid was extracted from the cultivated bacteria using officially provided manual by Saint-Bio company (Bacmid Extraction Kit 10123.100).
Cell culture and recombinant baculovirus production
Insect Spodoptera frugiperda clonal isolate 9 (Sf9) cells were cultured at 27℃ either adherently or in suspension. Specifically, the production of baculovirus in P1 and P2 generations was conducted using adherent culture, while the production in P3 generation was done in suspension culture. The culture medium we used were SIM SF Expression Medium purchased from Sino Biological company. For obtaining the P1 generation recombinant baculovirus stocks, the bacmid was transfected into the Sf9 cells in 6-well plate using LipoInsect™ Transfection Reagent (Beyotime Biotechnology) as described in the manufacturer’s protocol. The P2 generation recombinant baculovirus stocks were produced by infecting adherent Sf9 cell culture with P1 generation viral stocks. The P3 generation recombinant baculovirus stocks were obtained by employing similar method but the culture was in suspension so as to generate large quantity of viral stocks for future large-scale usage.
Extraction of the viral DNA
Viral DNA was extracted using the Rapid Viral DNA Extraction Kit (Aidlab Biotechnologies Co., Ltd; www.aidlab.cn), which is noted for its ease of use and reliability. Although the officially provided protocol is clear, we adapted it for extracting baculovirus DNA as described in the following.
Step 1: Invert the tube containing the harvested viral particles to ensure thorough suspension. Transfer 500 µL from the pre-harvested viral stock into a 1.5 mL Eppendorf (EP) tube for viral DNA extraction.
Step 2: Add 200 µL of the viral stock to an EP tube labeled with Preparation Tube and allow it to equilibrate to room temperature for at least 10 minutes. Reserve the remaining 300 µL for future use if necessary. Add 400 µL of Lysis Buffer DLB to the Preparation Tube and vortex thoroughly for complete mixing. Incubate the tube at room temperature (15–25°C) for 10 minutes, with additional mixing at the 5- and 10-minutes intervals.
Step 3: Add 450 µL of the anhydrous ethanol (below 25°C) to the Preparation Tube and vortex thoroughly. If the ambient temperature exceeds 25°C, pre-cool the ethanol before adding. Pre-cooling can be achieved by placing the ethanol on ice.
Step 4: Load the mixture from the Preparation Tube into an Adsorption Column in two 600 µL batches. Place the Adsorption Column into a Collection Tube and centrifuge at 12,000×g for 1 minute. Discard the liquid in the Collection Tube.
Step 5: Add 500 µL of Deproteinization Solution RE to the Adsorption Column, centrifuge at 12,000×g for 30 seconds, and discard the waste liquid.
Step 6: Heat the Elution Buffer (EB) in an EP tube to 70°C in a heatblock. This will be used in Step 9.
Step 7: Add 500 µL of Wash Buffer (WB) (ensuring ethanol has been added first) into the Adsorption Column, centrifuge at 12,000×g for 30 seconds, and discard the waste liquid. Repeat this wash step with another 500 µL of WB.
Step 8: Place the Adsorption Column back into the empty Collection Tube, centrifuge at 12,000×g for 2.5 minutes to remove excess WB so that to minimize the inhibitory effect of residual ethanol.
Step 9: Transfer the Adsorption Column to a new EP tube labeled with “DNA Tube”. Add 40 µL of pre-heated Elution Buffer (EB) to the bottom of the Adsorption Column and let it sit at room temperature for 1 minute, then centrifuge at 12,000×g for 1 minute. Reapply the solution obtained from the Adsorption Column, centrifuge at 12,000×g for 1 minute and mark the date on the ‘DNA tube’ (e.g., “24.7.1 Viral DNA”).
Preparation of the control PCR product for constructing the standard curve
We utilized the viral DNA extracted as described above as a template for PCR to produce the gene specific 180-bp fragment. Establishing a standard curve is essential for accurate viral titer determination, requiring the preparation of a gene specific PCR product with high purity.
Based on our extensive trials, we noted that the control PCR products should be freshly prepared for each experiment rather than using leftover preparations. Gene specific primers were designed not only for amplifying the control PCR product but also for performing the qPCR reaction. There are no stringent criteria for primer design other than ensuring they yield a PCR product of 180-bp.
For conducting the PCR using the Easy Taq system, the viral DNA extracted earlier was served as the template. For instance, in the case of our His.Def virus, we adhered to the PCR parameters including: Template – Extracted viral DNA; Primers – CDq-Fw, CDq-Rv; Annealing temperature – 58°C; Extension time – 8 seconds. The reliability of the PCR product was verified by running gel electrophoresis and followed by sequencing. Proceed to PCR product purification if the product band appeared single and corresponded to the expected size of 180-bp.
For PCR product purification, we utilized the SanPrep Column PCR Product Purification Kit purchased from Sangon Biotech (Shanghai) Co., Ltd. And we slightly modified it to better fit our usage. Despite the PCR product being only 180-bp in size, it has been observed that omitting additional isopropanol does not impact purification efficacy. The detailed procedures are as below.
Step 1: Transfer the PCR reaction mixture to an EP tube, add 5 volumes of Buffer B3 and thoroughly mix. This mixture was transferred to the Adsorption Column which was placed in a Collection Tube provided by the manufacturer. Centrifuge at 8,000×g for 30 seconds and discard the liquid from the Collection Tube. Add 500 µL of Wash Solution, centrifuge at 9,000×g for 30 seconds, and discard the liquid from the Collection Tube, and repeat this wash step once more. Centrifuge the Preparation Tube at 9,000×g for 1 minute to remove any residual liquid.
Step 2: Transfer the Adsorption Column to a new clean 1.5 mL EP tube. Add 50 µL of Elution Buffer to the center of the bottom of the Adsorption Column, let it stand at room temperature for 1 minute, then centrifuge for 1 minute to elute the DNA into the EP tube.
Step 3: Measure the concentration of the purified PCR product using Thermo NanoDrop 1000. Accurate concentration data is critical; therefore, for reliability, perform two measurements: first, establish a Blank measurement; then conduct the initial measurement, followed by a second measurement. If the results of the two measurements are similar, average them to obtain the final concentration.
Step 4: Record the concentration (denoted as "con") of the purified control PCR product in ng/µL for obtaining the molar concentration of the purified PCR product. This control PCR product of known concentration will be used for subsequent qPCR analysis.
Preparation of qPCR reaction samples
To generate the standard curve, the purified PCR products of known concentration was subjected to 1x, 101x, 102x, 103x, 104x, 105x and 106 dilutions. The resultant diluted DNA was used as the template for qPCR with three replicates for each dilution. Also prepare three replicates for each test viral DNA sample to be quantified. Prepare 10 µL of qPCR reaction mixture according to Table S2. Proceed to qPCR using the program parameters listed in Table S3.
Analyzing the qPCR data
Copy the generated qPCR data file from the instrument-connected computer to our own computer for analysis18. Ensure that Bio-Rad CFX Manager software has already been installed on our computer. Directly double-click the qPCR data file (with the .pcrd extension). The file will automatically be recognized and opened by Bio-Rad CFX Manager. Click on the Quantitation Data tab to view the qPCR data. The Target column displays the reaction names (defined by us when stopping the program), and the Threshold Cycle (Ct) column corresponds to the Ct values, indicating the template quantity in each sample. From the Target column to the Threshold Cycle column, drag to select all the necessary data. Right-click and choose "Copy" or press Ctrl + C to copy the data. Then, paste the data into Excel.
After appropriate editing, start to construct the standard curve. For the standard curve, use the data from the control group, which was 10x serially diluted. First, calculate the molecular weight of the control PCR product. To do this, open the Sequence Manipulation Suite website, which has a tool for this purpose. We can find the website by typing "Sequence Manipulation Suite DNA Molecular Weight" in the browser search bar, or by going to the URL (https://www.bioinformatics.org/sms2/dna_mw.html).
After opening the website, enter the sequence of the control PCR product into the input box, select "double" and "linear" for the parameters, and click Submit. A pop-up window will appear showing the molecular weight calculation result, denoted as MW, with the unit Da. Based on the concentration of the control PCR product from the previous content (denoted as con, in ng/µL), proceed with the following calculation. First, calculate the Avogadro’s number of double-stranded DNA molecules in the undiluted control PCR product (100 group, or say cont-0 group), denoted as Num:
Next, calculate the molecule numbers for other dilution groups and create the following table. Then, generate a trendline:
Based on the generated trendline, we can now proceed to solve the titers of the test viral samples (Fig. S3). Here is an example: a) Using the standard curve, substitute the Ct value of the test viral DNA sample to determine the log value of the test viral DNA molecule number. b) Then, using base 10, calculate the molecule numbers in 1 µL of the test viral DNA by taking the antilogarithm (exponential). c) Determining the number of test virus DNA molecules in 200 µL of virus solution, then converted it to the number of the test viral DNA molecules in 1 mL of viral stock (pfu/mL), namely, the titer of the test viral stock.
Thus, the titers of the two test groups in this experiment have been determined, with the unit being pfu/ml, which can be considered as the number of viral particles per milliliter. The viral titers measured by different methods may vary and reflect different aspects of the objective reality. When using this data, it is essential to be aware of its limitations and to ensure reproducibility by adhering to standardized protocols.
Ethics statement
The study in this article did not involve any trials on humans or animals.
Competing interests
The authors declare no conflict of interests.
Additional information
Correspondence and requests for materials should be addressed to J.R.P. This work was partly supported by the Hangzhou Chengxi Sci-tech Innovation Corridor Management Committee.
Author Contribution
FD did the investigation and data analysis and wrote the original draft. JYW helped with methodology and data analysis and editted the manuscript. HS helped with methodology and supervision. JRP conceived the project and funding acquisition and was responsible for project administration and supervision, and finalized the manuscript.
Acknowledgements
We thank Drs Jun Chen, Shuyi Zhao, Li Jan Lo and all members in the lab for their valuable suggestions.
Data Availability
Data is provided within the manuscript or supplementary information files. All data described here will be provided upon request to the corresponding author.
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No competing interests reported.