RespiFinder: a new multiparameter test to differentially identify fifteen respiratory viruses

Abstract

Broad-spectrum analysis for pathogens in patients with respiratory tract infections is becoming more relevant as the number of potential infectious agents is still increasing. Here we describe the new multiparameter RespiFinder assay, which is based on the multiplex ligation-dependent probe amplification (MLPA) technology. This assay detects 15 respiratory viruses in one reaction. The MLPA reaction is preceded by a preamplification step which ensures the detection of both RNA and DNA viruses with the same specificity and sensitivity as individual monoplex real-time reverse transcription-PCRs. The RespiFinder assay was validated with 144 clinical samples, and the results of the assay were compared to those of cell culture and a respiratory syncytial virus (RSV)-specific immunochromatography assay (ICA). Compared to the cell culture results, the RespiFinder assay showed specificities and sensitivities of 98.2% and 100%, respectively, for adenovirus; 96.4% and 100%, respectively, for human metapneumovirus; 98.2% and 100%, respectively, for influenza A virus (InfA); 99.1% and 100%, respectively, for parainfluenza virus type 1 (PIV-1); 99.1% and 80%, respectively, for PIV-3; 90.1% and 100%, respectively, for rhinovirus; and 94.6% and 100%, respectively, for RSV. Compared to the results of the RSV-specific ICA, the RespiFinder assay gave a specificity and a sensitivity of 82.4% and 80%, respectively. PIV-2, PIV-4, influenza B virus, InfA H5N1, and coronavirus 229E were not detected in the clinical specimens tested. The use of the RespiFinder assay resulted in an increase in the diagnostic yield compared to that obtained by cell culture (diagnostic yields, 60% and 35.5%, respectively). In conclusion, the RespiFinder assay provides a user-friendly and high-throughput tool for the simultaneous detection of 15 respiratory viruses with excellent overall performance statistics.

FIG. 1.

Overview of the RespiFinder technology. A one-step RT-PCR is performed with specific primers for all targets (steps 1A and 1B). Subsequently, an MLPA reaction is performed with MLPA probes specific for all targets (steps 2, 3, and 4). An MLPA probe consists of two oligonucleotides: one synthetic oligonucleotide and one M13-derived oligonucleotide. The synthetic oligonucleotide contains a universal forward priming site, and the M13-derived oligonucleotide contains a universal reverse priming site. In addition, the M13-derived oligonucleotide contains a unique stuffer sequence. The length of this stuffer sequence is specific for each probe and varies between the different probes. The length of the MLPA probe is the combined length of both oligonucleotides. This length is unique for each probe due to the specific stuffer sequence. The two oligonucleotides hybridize specifically to the target adjacent to each other. Subsequently, the two oligonucleotides are joined by ligation. The ligated oligonucleotides are amplified by one universal primer set. After amplification, the MLPA reaction is analyzed by electrophoresis (step 5). Each MLPA probe can be discerned due to its specific length. Recently, the protocol has been further optimized by combining the probe ligation (step 3) and probe amplification (step 4) in one reaction.

FIG. 2.

Overview of the RespiFinder probes and their length. nt, nucleotides.

FIG. 3.

Detection of the IAC in spiked serial virus dilutions on a 2.5% agarose gel. RespiFinder assay analysis was performed with fivefold serial dilutions of the supernatant of a PIV-1 culture. Lanes: 1, undiluted; 2, diluted 5¹; 3, diluted 5²; 4, diluted 5³; 5, diluted 5⁴; 6, diluted 5⁵; 7, diluted 5⁶; 8, blank. Each dilution except the blank was spiked with the IAC.

FIG. 4.

Results of the RespiFinder assay with two clinical samples which were analyzed by capillary electrophoresis. Sample 1 represents a clinical sample with a rhinovirus (Rhino) infection. The electropherogram shows a signal at the position of the rhinovirus probe and the IAC. Sample 2 represents a clinical sample with no viral infection. Only the IAC signal was detected in the electropherograms. Contr, internal amplification control.