Development of Traceable International Standard Materials Using Sequence-Customized Lentivirus, Modeled on SARS-CoV-2 Nucleic Acid Amplification Tests, and Validation through a Pilot External Quality Assessment.

Background: International standardization and quality control of nucleic acid amplification tests (NAATs) require traceable, full-process control materials aligned with international standards. We proposed a protocol for producing sequence-customized, traceable full-process controls, validated for stability and utility through a pilot external quality assessment (EQA) using SARS-CoV-2 NAATs as a model.

Methods: The concatenated SARS-CoV-2 NAATs target sequence was synthesized as a DNA fragment. A recombinant lentiviral material was generated using the Lentiviral High Titer Packaging Mix (pLVSIN-CMV Neo Vector, Takara Bio). Stability was evaluated after one freeze-thaw cycle at -80°C followed by incubation at 4°C or 25°C for seven days. The reference SARS-CoV-2 reverse transcription-quantitative PCR was standardized internationally using the WHO international standard material (National Institute for Biological Standards and Control). A pilot EQA of SARS-CoV-2 NAAT was conducted in 31 laboratories using high, low, and negative lentiviral materials.

Results: The 106-fold dilutions (7.41 x 103 IU/mL as low-concentration) in 10% FBS/DMEM of the original sequence-customized lentiviral materials were stable for seven days at 4 °C after freeze-thaw. Thirty laboratories reported positive results for high-concentration material, while 27 reported positive results for low-concentration material. GeneXpert (eight laboratories), with Ct values ranging from 22.6-26.7 for high and 32.5-37.0 for low-concentration. One laboratory using FilmArray reported negative results for all materials.

Conclusions: The sequence-customized and traceable lentiviral materials demonstrated suitable characteristics for EQA. The EQA results emphasize the importance of NAAT international standardization. These materials would help with NAAT international standardization, internal quality control, and EQA in individual laboratories.