Abstract
The Hologic Panther Fusion® Open Access™ functionality allows implementation of laboratory-developed tests (LDTs), with fully automated sample extraction, real-time PCR, and result interpretation. We report the development and validation of a multiplex LDT for norovirus G1, norovirus G2, and rotavirus from stool samples on this system. The LDT was optimized for primer and probe sequences, salt concentration, and PCR annealing temperature. Reproducibility of the PCR and extraction process was assessed. Performance of the multiplex LDT assay was evaluated with external quality assessment (EQA) samples and compared to a commercial multiplex assay (Allplex™ GI-Virus Assay, Seegene) in clinical samples. Salt concentrations and annealing/extension temperature were optimized to 4 mM MgCl2, 70 mM KCl, 20 mM Tris, and 60 °C, respectively. The user-prepared part of the LDT PCR mix (containing salts, probes, and primers) was stable for ≥ 11 days onboard the instrument. We observed reproducible results of PCR and the extraction process. The LDT had a sensitivity comparable to or greater than the commercial Allplex™ assay and showed excellent linearity. Forty-five EQA samples yielded the expected result with the LDT. There was 100% concordance between LDT and Allplex™ results in 160 clinical samples. Results from the suspension and direct swab stool sample preparation methods were highly concordant in the LDT. We report the successful development and validation of a multiplex PCR LDT for detection of norovirus G1, norovirus G2, and rotavirus from stool samples on the Panther Fusion® system.
Highlights
Rotavirus and norovirus are highly contagious viruses that cause acute gastroenteritis leading to severe diarrhea and vomiting [1, 2]
Polymorphisms present in more than 5% of the aligned norovirus G1 sequences and two additional polymorphisms detected in the sequences of clinical norovirus G1 positive samples were considered in the probe design
Since the maximal fluorescence with 0.2 μM probe was sufficiently high, this probe concentration was chosen for further assay development
Summary
Rotavirus and norovirus are highly contagious viruses that cause acute gastroenteritis leading to severe diarrhea and vomiting [1, 2]. Diarrhea is a leading cause of life expectancy reduction in all age groups [3], accounting for approximately 1.3 million deaths in 2015, including approximately 500,000 deaths in children < 5 years old [3, 4]. Norovirus accounts for 18% of all acute gastroenteritis leading to diarrhea and vomiting [5]. Norovirus causes 677 million cases of acute gastroenteritis in all ages, leading to approximately 214,000 deaths annually [6]. In infants and children under 5, rotavirus is the leading cause of severe diarrhea, accounting for more than 258 million episodes of diarrhea in 2016 [7]. Rotavirus vaccination has reduced rotavirus-related death since 2004, rotavirus infections still caused approximately 128,500 deaths worldwide in children < 5 years in 2016, accounting for 29% of all diarrhea-related death in this population [7]
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