Development of an identity test for COVID-19 mRNA vaccines using SARS-CoV-2 NAT standard

Highlights

• •mRNA vaccines can prospectively influence a major part of global public health.
• •RNA identity test in mRNA vaccines is a critical factor for quality control.
• •The qPCR method is a valuable tool for RNA detection in mRNA vaccines.
• •SYBR Green qPCR offers rapid RNA testing.
• •The method reduces reliance on proprietary reagents.

Abstract

Despite the development of messenger ribonucleic acid (mRNA) vaccines for the infectious novel coronavirus 2 (SARS-CoV-2), further research on test methods is required to ensure their quality as well as rapid and effective approval for release to the market. During the current national lot release testing, identity tests cannot be conducted on other products using primers, probes, and in-house reference materials provided by the manufacturer and specific to one vaccine, because their sequences do not match. When key reagents and reference materials are dependent on the manufacturer in this way, difficulties in national lot release approval—which serves as an additional step for the government to verify product quality—arise if the manufacturer does not provide them. In this study, we aimed to develop a quantitative polymerase chain reaction (qPCR) assay by using commercially available nucleic acid amplification test (NAT) reference material and a dye instead of a probe along with primers that were newly designed in this study. It can be applied to both vaccines. This study suggests a test method that can be applied when the in-house reference standard for the identity test, a major step to confirm the quality of vaccines, is not secured.

1. Introduction

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), led to the rapid development of messenger ribonucleic acid (mRNA) vaccines, but also highlighted the need for the development of new testing methods to ensure the quality of these vaccines [1]. However, for quality control of new platforms like mRNA vaccines, there are still, even after the end of the pandemic, no officially recognized testing methods listed in pharmacopeias such as the United States Pharmacopeia (USP) or European Pharmacopoeia (EP).

In Korea, the quality of each lot of biological products, such as vaccines, is confirmed by the national agency, considering the public safety prior to market release [2]. The tests required for lot release of vaccine are classified into general (appearance, pH measurement), product-specific (identity, potency, content, and purity), and safety (sterility, endotoxin) tests [3]. Among these, the identity test is a major product-specific test [3]. The identity test of mRNA vaccines involves verifying the sequence of mRNA. This test is very important in mRNA vaccines because the mRNA sequence determines the production of antigenic proteins associated with neutralizing antibody titers and affects the stability of the drug product.

To confirm mRNA sequences, nucleic acid testing (NAT) is employed as a reliable molecular diagnostic approach [4]. Among various nucleic acid testing methods, quantitative polymerase chain reaction (qPCR), loop-mediated isothermal amplification (LAMP), and Sanger sequencing are commonly utilized for the detection and analysis of specific nucleic acid sequences. LAMP offers rapid amplification under isothermal conditions without the need for sophisticated thermal cycling equipment, making it suitable for point-of-care applications [5]. Sanger sequencing, while regarded as the gold standard for sequence determination due to its high accuracy, is time-consuming, labor-intensive, and less sensitive for detecting low-abundance targets [6]. In contrast, qPCR combines the advantages of high sensitivity, specificity, and quantitative capability with relatively fast turnaround time. It enables real-time monitoring of amplification, allowing not only for detection but also for precise quantification of target mRNA levels, which is particularly useful in gene expression studies and clinical diagnostics [7]. Due to these features, qPCR is often considered the method of choice for accurate and reproducible quantification of nucleic acids.

“Standard materials” are reference substances used for the testing or analysis of biological products, including vaccines and pharmaceuticals. They are essential for ensuring accuracy and consistency, particularly in quantitative and qualitative assays such as NAT and immunoassays [2]. Reference standards can be classified as international, regional, national, and in-house reference standards. In-house reference standards, also referred to as working standards, serve as secondary reference materials. These standards are particularly useful in routine quality control testing, especially when international or national reference standards are not available or are in limited supply [8].

For national lot release approval by the Ministry of Food and Drug Safety (MFDS), Korea, quantitative reverse transcription PCR (qRT-PCR) or Sanger sequencing is conducted as an identity test for mRNA vaccines using in-house reference materials and specific primers provided by each manufacturer.

Modified nucleosides, such as pseudouridine, or unnatural modifications, are used in mRNA vaccines to reduce inflammatory effects and enhance in vivo stability [9]. Even when targeting the same antigen, the nucleotide sequence of vaccines may vary between manufacturers. Therefore, the primers and probes currently used for the identity testing required for national lot release approval are specific to each vaccine and its corresponding in-house reference material. If the manufacturer’s in-house reference materials for each vaccine for major quality tests, including identity testing, are not readily available, it could create a blind spot in quality assessment. In public health emergencies such as the COVID-19 pandemic, it is essential to establish testing methods that are independent of manufacturers and capable of providing rapid yet highly accurate results for the quality assessment of mRNA vaccines. This study aimed to develop a qPCR assay as an identity test method for mRNA vaccines using NAT reference material and newly designed primers instead of the manufacturer’s reference materials and primers/probes.

2. Materials and methods

2.1. Vaccines and reference material

The COVID-19 mRNA vaccines, COMIRNATY and SPIKEVAX, were provided by Pfizer Pharmaceutical Korea Ltd. and Moderna Korea Ltd., respectively.

The NAT reference material, the SARS-CoV-2 S gene (CRM No. 111-10-513), was purchased from the Korea Research Institute of Standards and Science (KRISS), which contained linearized plasmid deoxyribonucleic acid (DNA) harboring the SARS-CoV-2 S gene; plasmids were provided as a suspension in a Tris-Ethylenediaminetetraacetate (EDTA) buffer.

2.2. Primer design

To design the primer sets, the mRNA sequences of vaccines targeting the SARS-CoV-2 S gene and the DNA sequence of the NAT reference material were compared. Six primer sets were designed considering the differences in sequences between the vaccines and the NAT reference material (Table 1).

Table 1.

Primers designed and used in this study.

Primer set	Primer name	Sequence (5′→3′)*	Amplicon size (bp)
Set 1	F1	CAACAAAGTGACACTKGCMG	400
	R1	GCATTYTGGTTGACCACRTC
Set 2	F2	GGAACMGGAAGMGRATCAGC	310
	R2	RAACARYCKRTACAGGTART
Set 3	F3	GACAARAACACCCARGARGT	190
	R3	CCRTAYTGYTTGATGAAGCC
Set 4	F4	TGGATGGARAGYGAGTTCMG	200
	R4	CCCTGRGGSAGATCMCGCAC
Set 5	F5	TTYAACGCCACCMGRTTYGC	290
	R5	CCARGCKATMACGCAGCCKG
Set 6	F6	GGAACMGGAAGMGRATCAGC	250
	R5	CCARGCKATMACGCAGCCKG

*K = G + T, M = A + C, S = G + C, R = A + G, Y=C + T.

Abbreviations: bp, base pair; F, forward; R, reverse; G, guanine; A, adenine; C, cytosine; T, thymine.

2.3. RNA extraction

The COVID-19 mRNA vaccines, COMIRNATY and SPIKEVAX, were thawed following the instructions provided by the manufacturer. RNA was extracted by automatic extraction. For the automatic extraction, DNA extraction equipment (Applied Biosystems) and a nucleic acid extraction kit (Thermo Fisher Scientific, Catalog # P/N 4466351) were used.

2.4. Reverse transcription PCR

Complementary DNA (cDNA) was prepared using the mRNA extracted from the vaccine and the amfiRiver cDNA Synthesis Platinum Master Mix (GenDEPOT, Catalog # R5600), following the procedures recommended by the manufacturer. For this purpose, a reaction mix (20 μL) containing 500 ng of mRNA, 1 μL of cDNA synthesis enzyme mix, 10 μL of cDNA synthesis 2× buffer mix, and PCR-grade water was prepared. The reaction mixture was incubated at 25 °C for 1 min (annealing step), 50 °C for 30 min (extension step), and 75 °C for 4 min (inactivation step).

2.5. Traditional PCR

For each PCR reaction, 1 μL of cDNA sample was generated using the mRNA of the vaccine, or 1 μL of NAT reference material was used as the template. The template was mixed with PCR master mix containing 10 μmol/L (μM) forward and reverse primers (1 μL each), 2 μL of 50 mmol/L (mM) MgCl₂, 0.5 μL of 20 mM dNTPs, 0.5 μL of Taq DNA polymerase (5 U/μL), 5 μL of 10× PCR buffer, and 39 μL dH₂O. A PCR cycle included the following stages: 95 °C for 2 min, 30 cycles of 95 °C for 0.5 min, 53 °C or 58 °C for 0.5 min, 72 °C for 1 min, and the final elongation step at 72 °C for 1 min.

2.6. Agarose gel electrophoresis analysis and Sanger sequencing

The electrophoretic analysis of PCR amplicons was conducted using a 1 % agarose gel at 100 V for 60 min and visualized using Synergy Brands (SYBR) Safe 10,000× (Life Technologies, Catalog # S33102), a safe nucleic acid staining solution.

For Sanger sequencing, PCR amplicons were sampled through the clean-up, reaction, and purification steps. The amplicons were cleaned using ExoSAP-IT (Thermo Scientific^TM, Catalog # 78200.200. UL); subsequently, 40 μL of each amplicon and 20 μL ExoSAP-IT reagent were mixed and incubated at 37 °C for 15 min, 80 °C for 15 min, and finally maintained at 4 °C. For the reaction step for sequencing, 10 μL reaction mix was prepared using 2 μL of 5× buffer and 1 μL of BigDye Terminator v3.1 in BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Scientific^TM, Catalog # 4337455), 5 μL of nuclease-free water, 1 μL of DNA amplicon, and 1 μL of forward (F) or reverse (R) primer (primer set 4). The reaction was performed using the following program: initial denaturation for 1 min at 96 °C, followed by 35 cycles of 96 °C for 10 sec, 50 °C for 5 sec, and 60 °C for 2 min. For pre-sequencing purification, 45 μL of S-adenosyl methionine (SAM) solution and 10 μL of Xterminator solution (Thermo Scientific^TM, Catalog # 4376487) were added to the sample mixtures to stop the reaction, and then mixed for 30 min using the mixer. Samples made for sequencing were analyzed using an ABI 3500xL Genetic Analyzer (Applied Biosystems, Catalog # 4440463).

2.7. qPCR and melting curve analysis

A qPCR was performed in triplicate using 96-well plates in a final reaction volume of 20 μL; the reaction mix contained 10 μL of AccuPower^ⓡ 2X GreenStar^TM qPCR Mater Mix (Bioneer, Catalog # K-6251), 1 μL of 10 μM F primer, 1 μL of 10 μM R primer, 4 μL of nuclease-free water, 4 μL of template DNA, and cDNA or NAT reference material (10 times diluted using nuclease-free water). The qPCR cycles included the step for melting curve analysis as follows: 50 °C for 2 min, 95 °C for 10 min, 40 cycles of 95 °C for 15 sec, 60 °C for 1 min, and 95 °C for 1 sec.

3. Results

3.1. Primer design and selection

In this study, we aimed to develop an identity test method using newly designed primers that are applicable to all NAT reference materials and vaccines. Traditional PCR and electrophoresis were conducted to identify the most suitable primer set among the six primer sets listed in Table 1; the results are demonstrated in Fig. 1. We standardized PCR conditions, such as the annealing temperature (53 °C or 58 °C), to optimize the melting temperature. The newly designed primer sets were expected to generate a 190–400 base pair (bp) amplicon. The electrophoretic analysis revealed clear positive bands of appropriate size, except in the reaction set using the set 2 primer; moreover, no dimers were detected. These results indicated that all primer sets, excluding set 2, were highly efficient and exhibited a low tendency for self-dimerization. In selecting the optimal primer set for our study, we prioritized those that produced shorter amplicons, which are generally recommended for reliable qPCR performance. This decision was made to ensure compatibility with qPCR analysis [10]. Based on the high amplicon band intensity and considering the evaluation method used in this study, primer set 4 was selected as the optimal primer set.

Fig. 1.

Primer sets used in PCR and gel electrophoresis. For the selection of suitable primers, PCR and gel electrophoresis assay were conducted using the cDNA synthesized from the mRNA sequence of the vaccine or NAT reference material and various newly designed primer sets at different annealing temperatures conditions: 53 °C (A) or 58 °C (B). Abbreviations: PC, positive control; Pf, Pfizer vaccine; Mo, Moderna vaccine; PCR, polymerase chain reaction; cDNA, complementary deoxyribonucleic acid; mRNA, messenger ribonucleic acid; NAT, nucleic acid amplification test.

3.2. qPCR assay for the identity of the target gene

In the qPCR assay, we used NAT as a positive control (PC) and distilled water (DW) as a negative control (NC) to confirm assay acceptance. As shown in Fig. 2, when the qPCR assay was performed using the newly designed primers, amplicons were generated in the NAT reference material and vaccine reaction sets, whereas NC showed no amplification. These results imply that the vaccine samples contained the target strain. In addition, statistical processing of 13 quantification cycle (Cq) values (excluding undetermined [UD] ones) calculated for the DW group, showed maximum, minimum, median, and average values of 39.601, 32.864, 34.250, and 35.615, respectively; a standard deviation (SD) of 2.403 was detected. Therefore, by applying –3 SD, the Cq value of 28.000 was set as the criterion for identity determination (Fig. S1).

Fig. 2.

Amplification curve of qPCR reactions. Representative amplification plot demonstrating the efficient and specific amplification of the target gene using primer set 4 designed in this study. qPCR assays were performed on three templates: PC (spike gene DNA), Mo (cDNA synthesized from Moderna mRNA vaccine), and Pf (cDNA synthesized from Pfizer mRNA vaccine). The results confirm the successful design and specificity of primer set 4. Abbreviations: qPCR, quantitative polymerase chain reaction; cDNA, complementary deoxyribonucleic acid; mRNA, messenger ribonucleic acid; PC, positive control; NC, negative control; Pf, Pfizer vaccine; Mo, Moderna vaccine.

3.3. Melting curve analysis to confirm the specificity

The melting curve analysis was performed to confirm the specificity of the primer set 4. In the positive control and sample reactions, single melting temperature (Tm) peaks were observed (79.72 ± 0.08 °C, 87.46 ± 0.08 °C, and 87.92 ± 0.07 °C). No specific Tm peaks were detected in the negative control, which demonstrates the specificity of primer set 4 (Fig. S2).

3.4. Sequencing and amplification analysis

To verify the specificity of primer set 4, sequencing was performed using this primer set. cDNA samples were prepared from each mRNA vaccine, and Sanger sequencing was conducted using the NAT reference standard as a positive control. The results showed complete concordance between each sample and the expected reference sequences (Fig. S3). These findings demonstrate the specificity of primer set 4.

4. Discussion and conclusions

The World Health Organization (WHO) presents various methods for vaccine quality verification through its guidelines; DNA template sequencing and qRT-PCR are recommended for identity testing for mRNA vaccines [11]. As mRNA vaccines contain modified nucleosides, the mRNA sequences of different products, even if they target the same antigen, are varied. Therefore, identity tests cannot be conducted if reference material and primers/probes specific to individual vaccines are not provided by the manufacturer. This issue motivates the present study.

In public health emergencies such as the COVID-19 pandemic, the urgent need for rapid vaccine distribution has often limited manufacturers’ ability to provide reagents—including primers and probes—for independent quality control testing by national regulatory laboratories. The method developed in this study is not necessarily intended for routine application under normal conditions. Rather, it is proposed as a contingency approach that can be employed when manufacturer—provided materials are unavailable, particularly in future pandemic scenarios involving novel mRNA vaccines. This approach could help regulatory authorities maintain independent testing capabilities for mRNA vaccines during emergency situations, thereby supporting more rapid and flexible quality control operations without relying solely on proprietary materials.

This study aimed to develop an identity test method based on qPCR for mRNA vaccines using the NAT reference material and newly designed primers applicable to two different mRNA vaccines. Candidate primers were designed by aligning three sequences, including sequences from vaccines and NAT reference materials, and the most appropriate set (primer set 4) was selected using PCR and gel electrophoresis. The best primer set was selected based on the band intensity observed in gel electrophoresis and the amplicon size, which was suitable for optimized qPCR conditions.

We established a qPCR method using the universal dye, SYBR Green, which was non-specifically intercalated in the DNA double-strand during amplification, allowing for quantification. The SYBR Green principle is less restrictive in specificity than probes, such as those used in Taqman—based PCR assays [12]. To ensure the accuracy of the method, a melting curve analysis was performed, which confirmed both the specificity of the SYBR Green assay and the designed primers. We performed the method ten times, and based on the repeated results, we established criteria for positive and negative determination for the target strain. To further verify the specificity of primer set 4, Sanger sequencing was performed, and the results once again demonstrated that primer set 4 possesses template specificity for both mRNA vaccines and the NAT reference material.

This study has several limitations. First, the qPCR assay was validated only using NAT reference material and two mRNA vaccines, and its performance with a broader range of products remains to be demonstrated. Second, no direct comparison was made with internationally recognized reference standards, which would further strengthen the reliability of the approach.

In conclusion, this study suggests a test method that can be applied when the in-house reference standard for the identity test, a major step to confirm the quality of vaccines, is not secured. Moreover, we hope that the method proposed in this study can serve as an effective approach applicable to other mRNA vaccines that may be developed in response to future unpredictable diseases.

Supplementary data

The following are the Supplementary data to this article: