Revolutionizing Respiratory Diagnostics: The Transformative Impact of Real-Time PCR and Multiplex Methods


Discover how real-time PCR and multiplex methods are revolutionizing the diagnosis of respiratory infections!

In an era where rapid and precise diagnosis is paramount for effective clinical management, molecular techniques, particularly real-time PCR, have revolutionized our approach to identifying viral respiratory tract infections (RTIs). These cutting-edge methods have not only augmented diagnostic sensitivity and speed but have also vastly expanded the array of viral agents we can detect simultaneously.

Transformative Impact on Diagnosis

Traditional diagnostic methods for viral respiratory tract infections (RTIs) have largely been replaced by molecular techniques, mainly real-time PCR or multiplex PCR with liquid array (bead-based) detection systems. These molecular methods offer high sensitivity, rapid results, and the capability to detect multiple viral agents simultaneously. This has significantly affected the clinical approach towards diagnosing RTIs. Clinicians are now more reliant on virological tests for a definitive diagnosis, given the capacity of these methods to identify 15 or more different respiratory agents in a single sample.

Rethinking Clinical Diagnosis

The advancements in molecular diagnostics have posed a significant challenge to traditional clinical diagnostic methods. It has become increasingly evident that clinicians may find it hard to pinpoint the etiological (causative) agent based solely on clinical symptoms and history. One key finding is that multiple viruses often co-exist and replicate in the respiratory tract, potentially contributing to the symptoms. This has altered the perception that RTIs are generally caused by a single agent, impacting both treatment strategies and epidemiological understanding.

The Nitty-Gritty of PCR Methods

Various kinds of reporter systems are used in real-time PCR, each having its unique advantages and limitations. These include hydrolysis probes, molecular beacons, and fluorescence resonance energy transfer probes. The molecular diagnostic systems can detect an expansive list of agents, including but not limited to influenza viruses, respiratory syncytial virus (RSV), human metapneumovirus, and human coronaviruses. Several systems also test for bacterial pathogens like Chlamydia pneumoniae and Mycoplasma pneumoniae.

The Obsolete and the Exceptional

The arrival of these advanced methods has rendered older techniques like virus isolation in cell culture, antigen detection via enzyme immunoassays, and conventional qualitative PCR largely obsolete for routine clinical use. However, there are exceptions. Direct Fluorescent Antibody (DFA) tests are still used due to their rapidity, specificity, and cost-effectiveness, despite lower sensitivity. Similarly, cell culture techniques may remain relevant for identifying unknown infectious agents and for specialized research needs.

Epidemiological Impact

On the epidemiological front, real-time PCR and related techniques have offered unprecedented insights into the seasonality of viral respiratory diseases. This is crucial for public health policies and strategies aimed at mitigating the impact of seasonal viral outbreaks.

Relevance of Multiple Infections

The ability to identify multiple agents is not just a technological feat; it has substantial clinical implications. Infections with multiple viruses have been associated with more severe clinical symptoms. For instance, the course of Respiratory Syncytial Virus (RSV) bronchiolitis in infants may be exacerbated if there is a concurrent infection with Human Rhinovirus (HRV) or Human Metapneumovirus (HMPV). A similar pattern of aggravated symptoms has been suggested for other viruses like Human Bocavirus (HBoV), Human Parechovirus (HPeV), and classical enteroviruses.


In summary, real-time PCR and other molecular diagnostic methods have fundamentally transformed our approach to diagnosing and understanding respiratory viral infections. These techniques offer rapid, highly sensitive, and multi-agent detection capabilities that go beyond merely identifying pathogens. They challenge the traditional reliance on clinical judgment for etiological diagnosis and have exposed the frequent occurrence of co-infections.


  1. Olofsson, S., Brittain-Long, R., Andersson, L. M., Westin, J., & Lindh, M. (2011). PCR for detection of respiratory viruses: seasonal variations of virus infections. Expert Review of Anti-Infective Therapy, 9(8), 615–626.