![]() Among the benefits of this design, a chimera allows concurrent testing of disparate target regions of a single pathogen or even different organisms and splitting targets between standards A and B enables control cross-validation, facilitating the distinction of control failure from test failure or success. Furthermore, the equally partitioning of target sites between standards A and B enables cross-validation of positive and negative controls, increasing the confidence in test results. This means that for each target used in the molecular test, standard A would act as positive control, while standard B would act as negative control, or vice-versa. In this design, all the target sequences of a molecular test are retrieved and split between groups A and B, which are then joined in tandem to form single chimeric sequences A and B. ![]() Here, we propose a new design strategy for reference standards that uses matched chimeric synthetic standards in accordance with the principle of A/B testing. The use of in vitro synthesis of RNA and DNA standards allows flexibility in control design and tailoring of controls to the diagnostic test and targets. This leads to delays in diagnosis, missed diagnoses and invalidation of correct test results. For example, if the test returns a negative result from the positive control, it could be because (i) the test failed, (2) the reference control failed or (3) a technical issue with the testing platform. However, this failure of either positive or negative controls is difficult to distinguish from the failure of the diagnostic test itself. In the case of RNA standards, the synthetic controls can also undergo degradation over time, and can be contaminated, confounding the interpretation of test results. Reference standards must be validated and proven fit-for-purpose before used in diagnostic tests. However, separate negative controls, without the target sequences, are also required to ensure the specificity of the diagnostic test. These can be used as positive controls to assess sensitivity of the molecular test undergoing evaluation. The recent advent of DNA synthesis enables the rapid development of reference standards as synthetic constructs representing target genetic material. Reference standards are required to validate the performance of any diagnostic test 1. This chimeric reference standard design approach offers extensive flexibility, allowing representation of diverse genetic features and distantly related sequences, even from different organisms. The chimeric A/B standards were assessed using the US Centres for Disease Control real-time RT-PCR protocol, and showed results congruent with other commercial controls in detecting SARS-CoV-2 in patient samples. This enables control and test failures to be distinguished, increasing confidence in the accuracy of results. This design enables cross-validation of positive and negative controls between the paired standards in the same reaction, with identical conditions. Accordingly, a target region that is present in standard A provides a positive control, whilst being absent in standard B, thereby providing a negative control. The chimeric standards constituted target regions for RT-PCR primer/probe sets that are joined in tandem across two separate synthetic molecules. Using a pair of chimeric A/B RNA standards, this allowed incorporation of positive and negative controls into diagnostic testing for the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). However, negative controls are also required to evaluate test specificity. Current reference standards typically represent target genetic material, and act only as positive controls to assess test sensitivity. These are used as controls, and allow measurement and improvement of the accuracy and quality of diagnostic tests. DNA synthesis in vitro has enabled the rapid production of reference standards.
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