Evaluation of various filter paper and reagent systems for the preservation of Newcastle disease virus RNA samples

Humans, animals and the environment are key factors in the emergence and spread of various infectious diseases, meaning that interplay between factors is complex, outbreak locations can be multiple and simultaneous, and many early-intervention modes must be weighed up simultaneously, making the rapid analysis of samples to identify causative viruses crucial (20). Molecular assays, such as real-time reverse-transcription (RT)-PCR, provide fast, sensitive and specific detection, and are now widely utilised in diagnostic laboratories to identify a range of viruses, including Newcastle disease virus (25). However, specialised laboratories are often located far from the site of specimen collection, resulting in shipment times that can span several days. Viruses with RNA structure are chemically unstable and vulnerable to degradation by environmental RNases, which can compromise the integrity of the sample. This degradation may hinder a laboratory’s ability to accurately analyse the specimen and provide a reliable diagnosis (10). Virus samples should be stored in virus transport media at low temperatures, ideally frozen at -80°C, for transportation. However, this necessitates the use of dry ice, which has been classified as a “dangerous good” by the International Air Transport Association. As a result, specialised packaging and shipping protocols are required, significantly increasing the cost of transporting samples, particularly from field sites or remote hospitals (19). Thus, there is a growing demand for techniques that can reliably preserve viral RNA at room temperature for extended durations. Such methods would simplify the logistics and significantly lower the costs associated with transporting clinical samples or isolates to diagnostic laboratories.

Several reagents designed to stabilise RNA, such as GenTegra-RNA (GenTegra, Pleasanton, CA, USA) and RNAshell (Imagene, Pessac, France), are effective in maintaining RNA integrity at room temperature. Another such reagent is RNAstable, developed by Biomatrica (San Diego, CA, USA). This product is distinct in its approach, as it is based on the principles of anhydrobiosis, a biological process employed by certain multicellular organisms to survive in a dehydrated state (5, 24). However, these systems require RNA to be pre-extracted, which can be impractical if samples are being sent from a field site or a laboratory with limited technical equipment (24).

Alternative methods for preserving RNA at room temperature have been explored, including the Flinders Technology Associates (FTA) card (GE Healthcare, Rydalmere, NSW, Australia) and the RNASound card (FortiusBio, San Diego, CA, USA). These are specialised filter papers embedded with chemicals that stabilise nucleic acids in clinical samples (4).

The RNASound card offers a simplified RNA extraction and elution process. Virus samples are inactivated upon application to the card’s discs and can be easily eluted by dislodging the discs and shaking them in RNase-free water (13). In contrast, the Whatman paper FTA card is primarily designed for long-term DNA preservation at room temperature, with the manufacturer advising that RNA be processed immediately upon arrival at the laboratory or stored frozen (9). However, RNA from avian influenza viruses can be preserved on FTA cards for up to five months at room temperature (2). Additionally, RNA from certain arboviruses, such as Zika, chikungunya and dengue viruses, can be successfully recovered from FTA cards after 28 days of storage.

In this study, the efficiency of the FTA card, RNAstable and RNASound RNA preservation systems and the degradation of RNA in two controls – free RNA and free virus – was assessed using the LaSota strain of Newcastle disease virus. These preservation techniques and the controls were tested under varying storage temperatures and incubation periods. Following each incubation condition, the preserved RNA was evaluated for integrity and compared to RNA after different incubation conditions based on multiple criteria. The evaluation was made with real-time reverse-transcription (RT)-PCR analysis and Sanger sequencing.

Preserving viral RNA during transportation is especially challenging when samples are collected from remote locations with limited access to specialised laboratories. Current methods require freezing samples at –80°C, which involves the use of dry ice, classified as a dangerous good. This results in high costs and complex logistics for shipping. The issue is particularly critical for RNA viruses, such as the enveloped Newcastle disease virus, which are vulnerable to degradation during transit times. In this study, we aimed to evaluate the effectiveness of the FTA card, RNASound card and RNAstable tube viral RNA preservation methods, with two controls (free RNA and free virus) for comparison, focusing on their ability to preserve Newcastle disease virus LaSota strain RNA. These preservation methods were tested under different storage temperatures and time intervals to simulate real-world conditions. Following each storage period, we assessed NDV RNA quality, integrity and titre through real-time RT-PCR analysis, fluorometric measurement and DNA sequencing of the partial fusion gene to determine the overall efficiency and reliability of each method.

Overall, all preservation methods for NDV virus or RNA and the controls demonstrated good performance at lower temperatures. However, differences in their preservation performance became more apparent with prolonged incubation periods and increased temperatures, as revealed through the real-time RT-PCR, fusion gene sequencing and fluorometric RNA integrity measurement, which provided criteria to assess RNA quality and which have been employed in similar studies (13, 24).

In the real-time RT-PCR analysis, we measured the differences in C_t values for each preservation method, and for the control samples (free virus and free RNA) across various time points and incubation temperatures and compared them to the C_t values of samples stored at –80°C at each time point. The paper-based preservation methods (FTA cards and RNASound cards) and the RNAstable liquid solution demonstrated excellent preservation performance during the first week of incubation, yielding ΔC_t values of approximately 2. The FTA card gave a smaller ΔC_t for all time points and all tested temperatures except at 56°C on days 14, 28 and 35, when the ΔC_t ranged from 3 to 4. The other paper-based preservation method (RNASound) was more consistent at all tested time points and temperatures except on day 35 at 56°C, when the ΔC_t was around 2. The liquid-based preservation method (RNAstable) showed interesting results, with great performance at the 14- and 28-day points (except at 25°C) and then total disappearance of the NDV RNA at all tested temperatures on day 35. Free NDV virus had a smaller ΔC_t on days 1 and 7 at all tested temperatures, while on days 14, 28 and 35, the ΔC_t was smaller for the lower temperatures of –20°C and 4°C. By day 35, both the FTA card and RNAsound experienced a decline in RNA quality, with ΔC_t values reaching nearly 4. Notably, RNASound exhibited a significantly elevated ΔC_t value even on the first day of the 4°C sample collection, likely because of technical errors during sample handling or poor storage conditions. Overall, FTA cards and RNASound performed well across all temperatures, which aligns with existing literature on advanced nucleic acid preservation techniques. For instance, RNA from the avian influenza virus can be stored at room temperature using FTA cards for up to five months (13), and RNA from the Zika, chikungunya and dengue arboviruses can be successfully recovered from FTA cards even after 28 days (8). Similarly, the RNASound card effectively preserved influenza virus RNA for approximately 28 days at room temperature (13). Other preservation methods, such as those using spin column techniques, have also kept RNA stable at room temperature, as evidenced by MERS-CoV studies. In these studies, the average starting C_t value at week 0 was 18.524 ± 0.84, increasing to 20.25 ± 1.82 at week 16, indicating a difference of approximately 1.73 between the baseline C_t and the value recorded 16 weeks later (1). When used in our experiment, both of these methods exhibited comparable ΔC_t values.

RNAstable demonstrated promising results in real-time PCR analyses; however, by day 35, it exhibited extreme ΔC_t values across all storage conditions, highlighting a significant drawback of this technique. This issue mirrors findings by Stevens et al. (23), who encountered similar challenges while stabilising HIV viral RNA. They noted that an incipient in the product formulation adversely affected the real-time PCR analysis, possibly because it quenched the fluorescent signal.

The controls employed in the real-time PCR evaluation performed as anticipated. The free RNA remained intact only on day 1, after which it completely degraded and did not yield any C_t values. This may be ascribed to the inherent instability of RNA compared to DNA and the presence of RNA-degrading enzymes (RNases) (10). In contrast, the free LaSota strain NDV exhibited low ΔC_t values, comparable to those of the preservation methods tested. This observation underscores the significance of nucleic acid stability, as lyophilisation is commonly utilised in vaccine production to preserve viral antigens and adjuvants, thereby extending their shelf life (18).

Degradation of RNA occurs gradually, making the evaluation of RNA integrity crucial for obtaining reliable gene expression data. Agilent Technologies has introduced a novel RNA quality assessment tool, the RNA integrity number (RIN), which standardises RNA quality control through a numeric scale of 1 to 10, where 1 indicates maximum degradation and 10 signifies complete integrity (17). In our study, we utilised RNA integrity and quality assessment, which is reported similarly to the RIN. The IQ method employs two unique dyes: one that specifically binds to large and/or highly structured RNA, and another that targets small, degraded RNA (14). Measurements were taken using the Qubit 4 machine with the RNA IQ assay kit under conditions and at time points consistent with those adopted for RT-qPCR. The RNA from the FTA cards scored perfect 10s for integrity across all conditions and time points, demonstrating the cards’ capacity to preserve RNA integrity. Although limited research has evaluated the RIN for FTA cards, some studies report successful PCR results in all samples extracted from FTA Classic and Elute cards (22). Additionally, another study noted a relationship between RNA stability and ΔC_t values, with gradual degradation observed over 30 days at 37°C, while no significant differences in RNA amounts were found for samples stored at 4°C and 25°C (12). Similarly, lymph node fine needle aspiration samples stored for up to 123 days produced both 500-base-pair and 1.5-kilobase-pair amplicons in PCRs with nucleic acid extracted from FTA cards, indicating high nucleic acid integrity (22). Also, Manswr et al. (15) and Moscoso et al. (16) demonstrated the preservation of infectious bronchitis virus RNA for 21 and 36 days, respectively, at room temperature using FTA cards. Collectively, these studies suggest that FTA cards effectively maintain RNA integrity over extended periods and varying temperatures.

Although RNA samples provided reliable results during RT-qPCR, they did not exhibit a detectable RIN, which may be attributed to technical errors, improper sample handling, inadequate storage conditions or issues with kit preparation. In contrast, RNAstable at –80°C maintained a RIN of 9.2 across all time points. However, at elevated temperatures, the RIN became undetectable. A related study evaluated RNA stability in RNAstable after 4.5 months and 11 months of storage at room temperature and 45°C compared to a control stored at –80°C. The results indicated high RIN values of 9.8 for the stabilised RNA over the shorter storage period and 9.5 for the control. While the RIN of the stabilised RNA decreased from 9.8 at 4.5 months to 8.6 at 11 months, it remained within the range of excellent integrity (24).

The controls in the present experiment behaved as anticipated, beginning with high RIN values above 7.8 on day 1 across all temperatures. Although there was a slight decline in RNA quality by day 35, values remained above 7.1. Notably, at the low temperature of –80°C, the RIN averaged nearly 9 for both samples. While temperature did impact RNA integrity, there was still some retention of intact RNA.

Sanger DNA sequencing has long been regarded as the gold standard for determining nucleic acid (NA) sequences, whether they are naturally occurring or synthetically produced (6). This method not only serves as a confirmatory assay with high diagnostic specificity but is also crucial for the initial development of molecular assays. High-quality NA sequence analysis is essential for monitoring the efficacy of these assays and their components, as well as for evaluating the preservation methods used to ensure clear sequencing results. It is important to recognise that the quality of the PCR assay used to generate the sequencing target – encompassing primer design and the optimisation of reagents and conditions – significantly influences the quality and quantity of the resulting DNA or complimentary DNA amplicons for sequencing analysis (21). In our study, the three methods produced optimal results much of the time, with at least seven out of nine samples matching the reference, demonstrating consistent outcomes across various temperatures and time points. This suggests that PCR and sequencing reliably assess the integrity of the preserved virus/RNA methods. However, one control, free RNA, exhibited similar preservation characteristics, while the free virus control was negatively impacted by high temperatures and extended storage times, yielding only three reference-matching samples out of nine. This highlights the importance of storage duration and temperature, fixatives in the substrate and other treatments (such as demineralisation) on the stability and quality of RNA when it is preserved in diagnostic materials (6, 7).

Despite some drawbacks associated with extended storage periods, higher temperatures and potential technical errors during the extraction process, all three commercial RNA preservation methods demonstrated satisfactory performance and met quality assessments. These methods offer a viable alternative to the challenging transportation of samples at ultra-low temperature, particularly in remote locations. They enable the shipping of samples at ambient temperatures while maintaining RNA integrity, facilitating a range of molecular assays.