Infectious pathogens have traditionally been used as positive controls for PCR-based diagnostic methods24. However, utilizing them presents potential risks to researchers, particularly regarding airborne diseases that may spread to the environment25. Additionally, in the development of real-time PCR-based diagnostics, plasmid DNA is typically used to generate a standard curve to determine the copy number of a target sample26. This plasmid DNA can also be used as a positive control in PCR14. In our study, we aimed to evaluate whether the RT step affects the detection sensitivity when using plasmid DNA as the template, despite the fact that plasmid DNA itself does not require the RT step, which is necessary for detecting infectious pathogens harboring RNA as the genetic material27. As the J assay is designed to detect RNA viruses, such as VHSV, the RT step for subsequent PCR amplification is included by default. In real-time PCR without RT reaction, the RT step is bypassed, and the assay directly proceeds to PCR amplification. In this scenario, the absence of an RT step ensures that only the plasmid DNA is amplified, allowing us to determine whether the RT step introduces any variability in sensitivity. Our results demonstrated that both real-time PCR without an RT step and real-time RT-PCR (with RT) exhibited comparable detection sensitivity levels, indicating that the RT step in real-time PCR did not significantly affect detection sensitivity, even when cpDNA was used as a template. Therefore, plasmid DNA is a valuable tool for use as a PCR-positive control for both DNA- and RNA-type pathogen gene detection.
A standardized method is crucial to determining the accuracy of the positive and negative results of PCR-based disease diagnosis. In the present study, the potential of the J assay as a standard was investigated, and the detection sensitivity of several real-time PCR-based diagnostic methods was assessed. Our results demonstrated that the detection sensitivity of the ddPCR and J assays was comparable, indicating that the J assay could detect one copy of the VHSV gene. Consequently, the J assay can be regarded as a standard for validating the detection limits of various real-time PCR-based diagnostic methods. As the cpDNA contains the J assay sequence (primer set and probe) for the standard, as well as primer sets and probes for the diagnosis of several diseases, analysis using the cpDNA should detect the same copy number as the J assay if it has the same detection sensitivity.
In this study, IHNV, African swine fever virus, V. cholerae, and WSSV were detected at the same dilution level as in the J assay. In contrast, the VHS Garver assay and SARS-CoV-2 method detected pathogens at relatively lower dilutions than the J assay, indicating a relatively lower detection sensitivity. This low detection sensitivity could be attributed to the influence of the primers and probes used in the VHS Garver and SARS-CoV-2 assays. Consequently, the method developed in this study can be employed to validate the detection sensitivity of primer sets and probes, thereby facilitating the development of PCR-based diagnostic methods.
Our study demonstrated that the J assay can be used as a standard reference method to assess the sensitivity of PCR assays for other pathogens, such as IHNV, ASF, and V. cholerae.
This approach can be useful for ensuring that new methods meet established criteria for sensitivity and specificity, which is essential for the development and adoption of a new diagnostic method.
Using cpDNA also allows for flexibility in adapting the assay to various pathogens, as it can be easily engineered to include different target sequences for multiple pathogens. Furthermore, this could be a useful tool for avoiding false-negative reactions caused by poor detection sensitivity. In addition, this approach offers the advantage of developing and validating a robust molecular diagnostic method without pathogenic agents. Researchers can easily access the database and obtain the target sequence once the nucleotide sequence of the target pathogen has been analyzed and uploaded28,29. Furthermore, artificial intelligence can even predict viral mutation sites30,31; utilizing artificial intelligence might accelerate the development of accurate molecular diagnostic methods in a much safer environment with a relatively low chance of exposure to infectious pathogens.
To extend the application of the J assay as a standard for PCR, the probes employed for the J assay were tested using five different fluorescent dyes. The FAM probe demonstrated the highest relative fluorescence unit values among the five fluorescent dyes, although the Ct values of all reactions were nearly identical in the J assay.
The probability of false-positive results during PCR is high because of contamination by the positive control and its amplified products7,32, which can lead to misdiagnosis. Therefore, establishing a system that can effectively prevent false-positive results caused by genetic contamination is crucial for the precise diagnosis of diseases caused by infectious agents. In this study, an additional 20-mer probe sequence (contamination indicator probe) was introduced next to an existing probe site within the cpDNA using gene synthesis to prevent genetic contamination. Notably, the cpDNA used as positive control DNA in IHN diagnosis could be detected using the contamination indicator probe, demonstrating its potential as an indicator of false positives caused by contamination of samples.
By incorporating a contamination indicator probe, we can minimize the risk of false positives by detecting contamination early, preventing it from affecting the assay results.
This enhancement ensures the reliability of the diagnostic test, providing more accurate and trustworthy results. This feature is particularly beneficial in high-throughput or multi-sample testing environments where the risk of contamination is high.
While the implementation of a contamination indicator probe adds to the cost of the assay, the overall approach can be more cost-effective compared to methods that require the handling of live pathogens and associated extensive safety measures. Our approach of using plasmid DNA with diagnostic error prevention functions as a positive control also reduces the expensive and complex pathogen acquisition processes, making it more accessible to a wider range of laboratories.
It is also important to note that while the contamination indicator probe helps to detect false positives and secure more accurate diagnostic results, achieving higher diagnostic accuracy, we also need to develop and apply effective tools to deal with major contaminants within the diagnostic site. The cpDNA has been constructed following a designated molecular-based diagnostic method by an international organization. However, the cpDNA requires consistent updates to align with revisions or the designation of new diagnostic methods.
In this study, we developed recombinant plasmid-based standards and provided a useful tool for validating the sensitivity of different molecular diagnostic methods using the J assay as a standard. Furthermore, an innovative approach for introducing additional probe sites was designed to enable accurate diagnosis and reduce false-negative and false-positive results, which is critical for effective disease management and control.
The establishment of the J assay as a standard provides a benchmark for evaluating other diagnostic methods. This standardization facilitates comparison and validation of methods, ensuring consistent and reliable results across different laboratories and settings. These improvements enhance the accuracy and reliability of diagnostic tests, supporting better disease management and control in various settings.
These improvements make our approach more suitable for real-world applications, particularly in environments where rapid and accurate diagnosis is critical.
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- Source: https://www.nature.com/articles/s41598-024-78654-2