Evaluation of a laboratory-developed test for simultaneous detection of norovirus and rotavirus by real-time RT-PCR on the Panther Fusion® system

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.

Metabolomic Laboratory-Developed Tests: Current Status and Perspectives

“…However, there are also more sophisticated and complex LDTs, such as omics tests, with which it is possible to measure a large number of analytes. Several LDTs have been published for the diagnosis of various diseases, including genetic disorders, cancer, infections, and other disorders [ 14 , 15 , 16 , 17 , 18 , 19 , 20 ]. Among them, research papers on metabolomics have been published that describe the early diagnosis of Parkinson’s disease [ 21 , 22 ].…”

Multiplex Detection of Rotavirus A, B, C, Norovirus GI and GII, Adenovirus, Astrovirus and Sapovirus in a Single PCR System in Stool Samples From Children With Acute Diarrhea

“…However, due to the limitation of the number of uorescent dyes and labeled probes, it is impossible to detect more pathogens in one tube of detection solution. There are multiple PCR tests developed in previous studies [15,16]. Bennett et al developed one-step multiplex real-time RT-PCR assay simultaneously detecting adenovirus, astrovirus, rotavirus and sapovirus in one tube which has reached the limit detection of uorescent quantitative PCR in one system [17].…”

“…However, these techniques are time-consuming and are low in sensitivity and speci city [2]. In order to detect common viruses in children with diarrhea diseases, the high throughput, multiplex real-time RT-PCR was developed for the detection of adenovirus, astrovirus, rotavirus or sapovirus from stool samples in previous study [15][16][17]. However, the detection capacity of this method is not large enough.…”

Diagnosis of Parkinson’s Disease by A Metabolomics-Based Laboratory-Developed Test (LDT)

“…Some LDTs are relatively simple tests that measure single analytes; while other LDTs, like omics tests, are complex and may measure a large number of analytes. Recently, several LDTs have been published for the diagnosis of various diseases, including genetic disorders, cancers, infections and different other pathological conditions [11][12][13][14][15][16][17]. In this work, the LDT comprising the latest advancements in metabolomics research was tested as an early diagnostic method for PD, which is difficult for diagnosis using available laboratory tests.…”