Application of Multiplex PCR Coupled with Matrix-Assisted Laser Desorption Ionization–Time of Flight Analysis for Simultaneous Detection of 21 Common Respiratory Viruses

Abstract

Respiratory infections continue to pose a significant threat to human health. It is important to accurately and rapidly detect respiratory viruses. To compensate for the limits of current respiratory virus detection methods, we developed a 24-plex analysis (common respiratory virus-mass spectrometry [CRV-MS]) that can simultaneously detect and identify 21 common respiratory viruses based on a matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry system. To evaluate the efficacy of the CRV-MS method, we used 102 samples that were confirmed positive for these common respiratory viruses. All tests using the CRV-MS method were effective, with no cross-reactivity observed with other common respiratory viruses. To confirm the usefulness of the CRV-MS method, we screened 336 nasal and throat swabs that were collected from adults or children with suspected viral acute respiratory tract infections using the CRV-MS method and consensus PCR/reverse transcription-PCR (RT-PCR) methods. Excluding four RNase P-negative samples, the CRV-MS and consensus PCR/RT-PCR methods detected respiratory viruses in 92.5% (307/332) and 89.5% (297/332) of the samples, respectively. The two methods yielded identical results for 306 (92.2%) samples, including negative results for 25 samples (7.5%) and positive results for 281 samples (84.6%). Differences between the two methods may reflect their different sensitivities. The CRV-MS method proved to be sensitive and robust, and it can be used in large-scale epidemiological studies of common respiratory virus infections. R espiratory infections continue to pose a threat to public health. A World Health Organization (WHO) report indicated that lower respiratory infections (LRIs) remain among the top five causes of death worldwide, killing 3.1 million people in 2012. In low-income countries, LRIs are the number one cause of death (http://www.who.int/mediacentre/factsheets/fs310/en/ index.html). Many pathogens, especially respiratory viruses, are associated with respiratory infections. Different pathogens may cause similar clinical presentations, whereas their treatment differs greatly. Thus, it is important to accurately and rapidly detect respiratory pathogens.For different respiratory viruses, the mean time to detection of conventional cell culture varies from 3 to 8 days. Because many respiratory viruses exhibit high mutation rates, traditional methods do not to provide complete and reliable etiological information. Pavia (1) indicated that better diagnostic tests can reduce antibiotic use and control nosocomial transmission. At present, molecular methods have been widely used to detect respiratory viruses. With respect to traditional methods, molecular methods have great sensitivity and a short turnaround time, and they can simultaneously detect multiple pathogens (2). This progress has enabled us to better understand the phenomenon of pathogen coinfection. For example, based on a number of different platforms, many multiplex methods have been applied in the d...

Respiratory RNA Viruses

“…Lastly, the recent coupling of multiplexed RT-PCR with matrix-assisted laser desorption/ionization timeof-flight (MALDI-TOF) mass spectrometry analysis has led to the development of a sensitive, high-throughput, and robust system for the simultaneous detection of 21 common respiratory viruses (169). The platform uses two 384-well plates for a capacity of 760 clinical specimens within a single working day for large-scale screening of respiratory virus infections.…”

<p>Simultaneous detection of eleven sexually transmitted agents using multiplexed PCR coupled with MALDI-TOF analysis</p>

“…The system comes with some considerable benefits of multiplexing and high throughput, and it has been applied successfully in detecting and confirming various kinds of microbes. [17][18][19][20][21][22][23][24] For each microorganisms included in the STI-MS assay, two different genes were selected as targets. If either of the genes were detected, the corresponding organisms were positive.…”

Simultaneous Detection of Key Bacterial Pathogens Related to Pneumonia and Meningitis Using Multiplex PCR Coupled With Mass Spectrometry

“…MassARRAY System (Agena Bioscience, Inc., San Diego, CA, USA), a detection platform combining matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) with endpoint PCR, has been successfully applied in the field of microbial detection (Syrmis et al, 2011 ; Li et al, 2013 ; Peng et al, 2013a , b , 2014 , 2016 ; Zhang et al, 2015 ). Taking advantage of the system's multiplex capability and high sample throughput, we developed and validated a bacterial pathogen-MS panel (BP-MS) to simultaneously screen 11 key bacterial pathogens associated with pneumonia and meningitis, including Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Moraxella catarrhalis, L. pneumophila, M. pneumoniae , and Bordetella pertussis .…”