Comparative Analysis of COVID-19 Gene Target Dropout/Failure Results using Thermofisher TaqPath COVID-19 Combo Kit and Nextstrain CoVariants Genomic Databases

Abstract

COVID-19 variants continue to infect thousands of people even though the end of the pandemic was announced on May 11, 2023. Nextstrain CoVariants (CoVariants) genomic databases provide detailed information about more than 31 variants of COVID-19 viruses that have been identified through genomic sequencing, showing the mutations they carry. Mutated viruses may yield a negative result for a gene target using a PCR test that has a positive COVID-19 test result. This negative gene target result is known as gene target dropout/failure, not a negative COVID-19 test result. The Centers for Disease Control and Prevention (CDC), World Health Organization (WHO), and CoVariants utilize gene target dropout/failure as a Variant of Concern (VOC) screening method until the genomic sequencing is performed. The Thermofisher TaqPath COVID-19 Combo kit (TaqPath), a real-time RT-PCR test, detects COVID-19 nucleic acids in respiratory specimens. TaqPath PCR COVID-19 target profile data was retrieved from the Tuskegee Health Disparities Diagnostic Center COVID-19 clinical laboratory. The results revealed an association between the TaqPath results date tested and CoVariants tracking announced and end dates for S gene target dropout/failure (SGTF). This study highlights the usefulness of TaqPath COVID-19 Combo Kit gene target profiles in monitoring and mitigating the spread of emerging COVID-19 variants, without genomic sequencing.

Introduction

COVID-19, a disease caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), was identified in December 2019 in Wuhan, Hubei province, China. Initially distinguished as a cluster of pneumonia cases with an unknown cause, this virus quickly spread worldwide. As a result, the World Health Organization (WHO) declared a Public Health Emergency of International Concern on January 30, 2020, and then a pandemic in March 2020 (Wilder-Smith & Osman, 2020).

Unprecedented public health measures, including lockdowns, travel restrictions, widespread testing and quarantine protocols, were implemented in response to the rapid spread of COVID-19 (Wibmer et al., 2021). The global healthcare systems, daily life, and economies of the world were significantly impacted by the pandemic. In response, scientists across the globe were mobilized, aiming to understand the virus and create effective treatments and vaccines. Vaccines were crucial to curbing the spread of the virus and decreasing its severity, along with public health measures (Baden et al., 2021).

Viruses are small infectious agents that can only multiply within the living cells of animals, plants, and bacteria. When a virus replicates within a cell, errors or mutations happen. Amicone, et al. (2022) discovered that SARS-CoV-2 mutated at a significantly high rate and that the Spike protein has the potential to adapt to new environments. Monitoring the emergence of new variants is important, as new variants may diminish the effectiveness of vaccines, and treatments, and clinical tests. Any mutation that occurs can potentially affect the ability to detect target genes in molecular biology technologies. Variants can only be confirmed via genomic sequencing, a laboratory method that determines the genetic makeup of an organism. Genomic sequencing can also determine changes in an organism caused by the mutation. However, this process is costly, time-consuming, and unavailable in clinical laboratories (Nicot et al., 2023). Molecular biology techniques such as polymerase chain reaction (PCR) detect specific genes in a virus. Changes in gene detection can signal a mutation. This study will explore gene dropout/failure for the Spike gene using the Thermofisher TaqPath (TaqPath) COVID-19 Combo kit to as a screening (proxy) for emerging variants.

According to the CDC, viruses such as SARS-CoV-2 continuously changed throughout the pandemic due to genetic mutations and viral recombination. These changes, occurring in the virus’ genome during replication, have produced variants that deviate from the original strain (Nicot et al., 2023). Throughout the COVID-19 pandemic, many variants of SARS-CoV-2 have been found in the United States and globally. The CDC monitors all variants of concerns (VOC) in the United States. A VOC designation includes factors such as increase in transmissibility, disease severity (e.g., increased hospitalization or death), significant reduction in neutralization by antibodies generated during previous infection or vaccination, reduced effectiveness of treatments or vaccines, and diagnostic detection failures. Mutations can take place within any of the SARS-CoV-2 genes (Amicone et al., 2022). Variants of Concerns have the ability to evade the immune system which allows the virus to be easily transmitted, creating a problem for everyone. Vaccinated individuals may not be fully protected due to the mutation on the spike protein, the area where antibodies bind. Figure 1 shows the genetic sequence of the original SARS-CoV-2 virus and the mutated B.1.1.7 with the 69/70 deletion. This deletion results in the Spike (S) target dropout/failure in the TagPath COVID-19 Combo kit, i.e., the S target will appear as negative.

Figure 1.

Genomic sequence of SARS-CoV-2 showing 69/70 deletion (Meng, 2021).

The TaqPath COVID-19 Combo Kit is a diagnostic tool used to detect nucleic acids from SARS-CoV-2, in nasal specimens («Thermo Fisher Scientific (2020a),» 2022). The combo kit is a multiplex real-time RT-PCR assay that the U.S. Food and Drug Administration granted an Emergency Use Authorization (EUA). The Combo Kit consists of the following three components which are necessary for the detection of SARS-CoV-2: 1) TaqPath COVID-19 Control, 2) the TaqPath COVID-19 Combo Kit Primer and Probe Mix, and 3) the TaqPath 1-Step Multiplex Master Mix (Thermo Fisher Scientific, 2020a). Figure 2 illustrates the structure of the virus, target genes, and its corresponding genome. Each of these assays target different regions of the SARS-CoV-2 genome: ORF1ab, N gene, and S gene. The Spike (S) gene is responsible for the virus attaching to the host cell. The Nucleocapsid protein (N) is the viral protein coat that surrounds the DNA or RNA of the virus. The ORF1ab region is the largest gene that contains the overlapping open reading frames that encode polyproteins responsible for viral transcription, replication, and other processes. Using a multi-target approach, this kit provides a robust response that helps minimize false negatives that could stem from the genetic variations in the virus (Corman et al., 2020).

Figure 2.

Structure of the virus, target genes, and its corresponding genome (Jamison, 2022).

The workflow of the TaqPath COVID-19 Combo Kit was designed to be efficient and handle high-volume testing. It starts with the extraction of RNA from clinical specimens through nasal swabs. The extracted RNA is then added to the real-time PCR reaction, which contains a master mix, primers, probes, and controls. Then a PCR amplification is done using an instrument that emits a signal when it detects the nucleic acid being amplified («Thermo Fisher Scientific (2020a),» 2022). The high sensitivity of the combo kit makes it possible to detect SARS-CoV-2 even if a person has a very low viral load. Also, using very specific primers and probes makes the likelihood of this assay cross-reacting with anything but SARS-CoV-2 low to none (Tahamtan & Ardebili, 2020). The ease of use and reliable results have made this kit an essential tool for public health laboratories and hospitals worldwide. Figure 3 shows the amplification of the S, N, and ORF1ab targets and S gene dropout/failure for the TagPath COVID-19 Combo kit results.

Figure 3.

Amplification of SARS–CoV-2 targets: A. all three targets present; B. only two targets present (Images from the THDDC Laboratory)

Usually detecting target dropout/failures is only observed in research laboratories. However, if clinical laboratories reviewed gene target values in addition to positive and negative results, possible mutations could be detected before genomic sequencing. Clinical laboratories do not report viral targets detected and many laboratories may not review the target profile. Reporting gene target value changes to state public health agencies would provide valuable information on emerging variants in real time.

This research builds on current knowledge by recommending specific ways to incorporate S-gene dropout data into public health strategies. Unlike past studies that examined lab or research settings, this study sheds light on how direct clinical lab results can inform public health decisions, potentially leading to more timely, targeted interventions during future outbreaks.

Research Question

Can the S-gene dropout/failure be used as a proxy to determine COVID-19 circulating variants using the TaqPathRT PCR Combo kit?

Hypothesis

There is an association between the Nextstrain CoVariants VOC date announced and ended and SGTF, coded in dichotomous variables (S Gene present or not present)

Literature Review

The COVID-19 virus is highly transmissible, leading to a rapid increase in the number of cases and confirmed infections worldwide (Control, 2021). Vaccination, diagnosis, isolation, and treatment were vital to controlling the spread of the disease. Test methodologies proved successful in detecting either the virus or proteins in patient samples. The PCR, considered confirmatory and the gold standard, detects viral genetic materials using known primers (gene code). The antigen test is a screening test that detects proteins produced by the virus. The PCR test can detect a small amount of the virus or viral particle; however, for an antigen test to be positive, a large amount of the virus must be present, usually in a symptomatic individual. Thus, one can have a negative antigen test and a positive PCR test (Corman et al., 2020).

Viral mutation affects testing methods’ ability to amplify targets. The “gene drop out” or “gene target failure” phenomenon can detect new variants of viruses (Bozidis et al., 2022). Particularly, changes in the S-gene at the 69–70del mutation (a 6-nucleotide deletion) resulted in the S-gene using the Thermo Fisher TaqPath COVID-19 Combo kit not to be detected. Because of the two other genes (N and ORF1ab), positive results were obtained. This pattern indicated that a new variant had emerged. The new variant was sequenced and labeled Alpha in November 2020. Other variants emerged that did not have the 69/70del. Later, a new variant, Omicron emerged with the 69/70 del resulting in the S-gene dropout/failure (Ghandhi, 2022). The del 69/70 mutation results in the S gene failure or a negative result using PCR tests is characteristic of the Alpha and Omicron (BA.1.1.529, BA.1, and BA.1.1) (Clark et al., 2022; Meng et al., 2021).

Samples investigated by McMillen et al., (2022) using the Taqpath RT PCR SGTF for the identification of the SARS-CoV-2 Alpha (B.1.1.7) and Omicron variants (B.1.1.529. BA.1, BA.4 and BA.5) showed a greater than 95% sensitivity and specificity. Specifically, the positive predictive value for SGTF was 98% for Alpha and 100% for Omicron. This study highlighted the accuracy of using SGTF as a rapid and accurate proxy for identifying variants in a clinical laboratory setting (McMillen et al., 2022). Therefore, the ThermoFisher TaqPath RT PCR Combo Kit that tests for the S, N, and ORF1ab genes fails to amplify the S gene with the del 69/70 mutation and can be used as a proxy for the Alpha and Omicron (BA.1.1.529, BA.1, and BA.1.1) variants.

The relationship between the presence of VOC and S gene target dropout/failure was reinforced by analysis. TaqPath results were obtained from all the samples with genomic data. Sequencing was performed on lab controls to confirm the results. When all the data was combined, it was determined that the S gene target failure was most commonly associated with the Alpha variant and with several Omicron subvariants (Clark et al., 2022).

Since December 2020, six VOCs have been named, Alpha, Beta, Delta, Gamma, Epsilon, and Omicron. Over 1,900 lineages (mutated variants) of SARS CoV-2 have been identified via genomic sequencing (McMillen et al., 2022). Alpha and Omicron share the del 69/70 mutation. As the pandemic progressed, Alpha, Beta, Gamma, and Epsilon were downgraded to variants of monitoring when Delta emerged in September 2021. Omicron entered late November and replaced Delta as the predominant variant (Clark et al., 2022). The amplification of the Spike gene (S-gene) in RT-PCR can be utilized to differentiate specific SARS-CoV-2 lineages over time (Link-Gelles et al., 2024). The S-gene, the divergent of the JN.1 lineage, has more than 30 mutations in the spike protein and is accounted for high infections (Link-Gelles et al., 2024). The S-gene target presence (SGTP) and SGTF or S-gene dropout/failure are known as the proxy for JN.1 compared to other lineages (Link-Gelles et al., 2024). Observing SGTP and SGTF are important as new variants of concerns emerge.

Methods

This research compared test results for SARS-CoV-2 using the ThermoFisher TaqPath COVID-19 Combo Kit with CoVariants Genomic Databases. The study aimed to evaluate the ability of the TaqPath assay to screen for circulating VOCs with gene target dropouts/failure in the S gene. To ensure the reliability and validity of the data, a cross-reference of the TaqPath test results with genomic sequencing data from the CoVariants databases was conducted. This study excluded sample results that were positive for all three targets (S, N and ORF1ab). The project received IRB approval (HPRC #082323) from Tuskegee University on August 29, 2023, to compare TaqPath target values obtained in Tuskegee University Health Disparities Diagnostics Center lab with genomics databases.

The TaqPath PCR COVID-19 target profile data was retrieved from the Tuskegee Health Disparities Diagnostic Center COVID-19 clinical laboratory. An analysis of the TaqPath date tested and negative gene result was conducted and compared to the CoVariants databases of VOCs date variant announced and ended. A total of 15,397 COVID-19 samples tested using the TaqPath method between February 9, 2021, and December 17, 2023, were assessed to identify results with gene target dropout/failures.

Excel spreadsheets that tracked the S gene target dropout, daily test results, date of test results, the VOC with the 69/70 deletion for the S gene, and dates CoVariants began tracking the VOC, and the date CoVariants stopped tracking the VOC were created. A Pearson’s chi-squared test to determine the association between the variables of concern, CoVariants VOC date announced and ended, and SGTF, coded to dichotomous variables (S gene present and S gene not present). The statistical tool, SPSS version 26.0, was used to analyze the data using Chi-square/Fisher’s exact test to assess the significance and correlation of the data, and p-values of <0.05 were considered to be significant.

Limitations

The study is limited to the use of the S-gene dropout/failure and information provided by the CoVariants Genomic databases and results from the Tuskegee Health Disparities Diagnostic COVID-19 lab at the time of the study. The study covers VOCs announced from January 1, 2020 to December 31, 2023 and test results from February 9, 2021 to December 31, 202

Results

The study analyzed the TaqPath date-tested result compared to the CoVariants (genome international database) VOCs date variant announced and ended to evaluate the S gene target dropout/failure in the test pool. COVID-19 samples (N=15,397) were tested using the TaqPath Combo kit method between February 9, 2021, and December 17, 2023. Of the sample pool (n=922) SGTF were selected for analysis: Analysis revealed Alpha, 20I, VI (68, 7.4%), Omicron 21K, BA.1 (168, 18.2%), Omicron 22A, BA.4 (377, 40.9%), and Omicron 22B, BA.5 (309, 33.5%), were present in the test sample pool. The laboratory results showed SGTF for the Alpha (20I, V1) variant between December 2020 and late September 2021 and the Omicron variants 22A (BA.4), 22B (BA.5), 22D (BA.2.75) and 21L (BA.1) beginning late 2022. The Alpha and Omicron variants were both prevalent during the testing periods. The Tuskegee Health Disparities Diagnostics Center lab data revealed that the SGTF testing technique helped highlight the prevalence of four (4) VOCs.

When analyzing the VOC using the TaqPath kit test to determine if the S gene was present or not present, Table 1 showed that Alpha, 20I, VI (8.8% present, 91.2% not present), Omicron 21K, BA.1 (20.8% present, 79.2% not present), Omicron 22A, BA.4 (6.9% present, 93.1% not present), and Omicron 22B, BA.5 (1.6% present, 98.4% not present) respectively. Table 1 shows the percentages of cases where the S-gene was present and not present. The data suggest that the TaqPath test indicated that 7.8% of the S gene were present while 92.2% were not present when compared to the Global VOC. Suggesting that there is a predominant presence of variants associated with SGTF.

Table 1.

Global VOC by TaqPath SGTF

	TaqPath_SGTF	SGene-Not Present
	SGene-Present
GlobalVOC	Alpha, 20I, VI	8.8%	91.2%
	Omicron 21K, BA.1	20.8%	79.2%
	Omicron 22A, BA.4	6.9%	93.1%
	Omicron 22B, BA.5	1.6%	98.4%
Total		7.8%	92.2%

The research question posed was whether the S-gene dropout/failure could be used as a proxy to determine COVID-19 circulating variants using the TaqPath RT PCR Combo kit. Additionally, the study hypothesized an association between the CoVariants VOC date announced and ended and SGTF, coded in dichotomous variables (S Gene present or not present). The results of the Chi-squared showed that the squared statisticx^2 (2)=56.568, Degree of freedom 3, corresponds to p < 0.001 which reinforces the usefulness for observing the frequency and spread of SARS-CoV-2 variants in real-time. Therefore, the null hypothesis was rejected with 99.9% confidence, as shown in Table 2, which shows strong evidence of an association between the CoVariants VOC date announced and ended and SGTF. The cross-referencing of the results of the TaqPath with the genomic sequencing data available in CoVariants disclosed the positive relationship between the S gene target dropout/failure to the VOC. S gene dropout was most often associated with Alpha, Omicron (22A, 22B) and Omicron 21K. The S-gene dropout/failure can be used as a proxy to determine COVID-19 circulating variants using the Taqpath RT PCR Combo kit. Understanding that the S gene dropout is a strong indicator of the presence of the SARS-CoV-2 variant means that clinicians can make better-informed decisions about treatment. This, in turn, can help in recovery and mitigate the further spread of the virus in the population.

Table 2.

Chi-Square Tests Global VOC by TaqPath RT PCR Combo kit

	Value	df	Asymptotic Significance (2-sided)
Pearson Chi-Square	56.568a	3	.0001
Likelihood Ratio	52.498	3	.0001
N of Valid Cases	922

^a0 cells (0.0%) have an expected count of less than 5.

The minimum expected count is 5.31.

The results revealed an association between the TaqPath date tested and ended and SGTF. Since p< .05, the test indicates that there is a significant relationship between the TaqPath combo kit and S gene target failure. The cross-referencing of the results of the TaqPath with the genomic sequencing data available in CoVariants disclosed the positive relationship between the S gene target dropout/failure to the VOC. S gene dropout was most often associated with Alpha, Omicron (22A, 22B) and Omicron 21K. The results validate the TaqPath as a reliable means for detecting SARS-CoV-2 variants. This is important for public health surveillance, as it allows for a more effective response to the pandemic.

Discussion

The study highlights the effectiveness of the TaqPath RT PCR Combo kit for screening SARS-CoV-2 variants through dropout/failures in the S gene target. The Alpha variant (B.1.1.7), one of the first variants of concern (VOC) identified, demonstrated a dropout/failure in the S gene target due to a deletion at amino acids 69/70 in the spike protein. The TaqPath Combo kit resulted in a «not detected» or «dropout» signal for the S gene, while it continued to detect the presence of the N gene and the ORF1ab gene, which indicated that a new variant was present.

Figure 4 represents the VOC timeline. The dates for October 2021 were associated with Omicron 22A, BA.4, which became the predominant variant in November 2021, while the Alpha, 20I, and VI variants had significantly decreased in prominence. Additionally, the Beta, Delta, and Gamma variants appeared between December 2020 and May 2022.

Figure 4.

Variant of Concern Timeline

The Tuskegee Health Disparities Diagnostics Center lab data revealed that the SGTF testing technique was useful in highlighting the prevalence of four (4) VOCs. The laboratory results showed SGTF for the Alpha (20I, V1) variant between December 2020 and late September 2021 and the Omicron variants 22A (BA.4), 22B (BA.5), 22D (BA.2.75) and 21L (BA.1) beginning late 2022.

Conclusion

This study conducted a comparative analysis of COVID-19 gene target dropout/failure results using the Thermo Fisher TaqPath COVID-19 Combo Kit and the Nextstrain CoVariants Genomic Database. The study highlighted the connection between S gene target dropout/failure, the phenomenon by which the S gene is not detected by some commonly used PCR COVID-19 test methods. It was established that S gene target dropout/failure serves as screening (proxy) for emerging variants of concern. This tool can provide a rapid assessment of how extensively SARS-CoV-2 is mutating in a given population.

Combining TaqPath screening with genomic sequencing provides a strong, effective method for detecting and monitoring variants of concern. TaqPath enables rapid, scalable, and sensitive screening and is particularly useful for the high-throughput detection needed in a public health emergency. Genomic sequencing, which takes more time, provides insightful detail at the molecular level. When the use two methods are used together, the capacity to identify potential variants is substantially increased, giving public health authorities a much more accurate and reliable picture of circulating VOCs in the communities.

This study underscores the utility of TaqPath COVID-19 Combo Kit gene target profiles in the surveillance of circulating VOCs to monitor and mitigate the spread of COVID-19 in the absence of genomic sequencing. Genomic sequencing is required to confirm the identity of the VOCs. Further investigation is warranted to determine the association between the N and ORF1ab genes target dropout/failures and VOCs.

Recommendations

Clinical laboratories could use gene target values to screen for emerging VOCs in patient samples. This information could be useful for physicians to provide better treatment plans and monitor recovery progress. Public health officials can use this information to compare genomic sequencing and data from water samples to determine outbreak hot spots and variants in real time.