High-throughput Sequencing Resolves Conflicting PCR Results to Confirm Suspected 2019-nCoV Infection
When a 65-year-old patient in Guangzhou developed pneumonia after potential contact with a COVID-19 case in January 2020, doctors hit a diagnostic wall: two different PCR tests gave conflicting results. One commercial kit labeled the throat swab negative (cycle threshold, or CT, values >40, meaning no detectable viral RNA), while a second kit showed weak positivity (CT values 31–38, indicating low levels of 2019-nCoV). For cases like this—where epidemiological links, symptoms, and imaging suggest COVID-19 but standard tests are unclear—high-throughput sequencing (HTS) proved to be a game-changer.
The Diagnostic Dilemma: Why PCR Isn’t Perfect
Fluorescence quantitative PCR (FQ-PCR) is the backbone of COVID-19 testing. It works by amplifying viral RNA and using fluorescent signals to measure how many “cycles” are needed to detect the virus (lower CT = more virus). But here’s the catch: not all PCR kits perform the same. Variations in design, sensitivity, and target genes (like ORF1ab, E, or N, which code for key viral proteins) can lead to inconsistent results—even for the same patient sample.
For this 65-year-old with a suspected exposure history and pneumonia, the conflicting PCR results left clinicians unsure. Was the patient infected? Or were the weak signals a false positive? To answer that, the team turned to meta-transcriptomic sequencing—a type of HTS that reads all genetic material in a sample, including viral RNA.
How HTS Settled the Case
Using the same throat swab that yielded mixed PCR results, researchers built a sequencing library and ran it on an Illumina NextSeq 550Dx machine. They analyzed the data with a rapid pathogen detection system (RPD-seq) designed to spot viruses quickly. The result? Ten unique genetic “reads” that perfectly matched the 2019-nCoV genome (GenBank accession MN908947.3). Crucially, these reads were spread evenly across the virus’s genes—including the ORF1ab, E, and N regions targeted by PCR tests. This distribution ruled out contamination (which would show clumped reads) and confirmed a weak but real infection.
Why This Matters for COVID-19 Diagnosis
China’s National Health Commission lists either positive FQ-PCR or viral gene sequencing as the gold standard for COVID-19 diagnosis. But as this case shows, PCR alone can leave gaps. HTS, while not practical for routine use (it’s costly, labor-intensive, and requires specialized equipment), offers a critical backup for ambiguous cases. For the 65-year-old patient, it turned uncertainty into clarity—ensuring proper treatment and infection control measures.
The Bigger Picture
The study, led by researchers from the Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University, and partners including the State Key Laboratory of Respiratory Disease and Macau University of Science and Technology, highlights a key lesson from the pandemic: no single test is perfect. When PCR results are unclear, sequencing can step in—if labs follow strict quality controls to avoid errors.
Wen-Da Guan and Li-Ping Chen contributed equally to the work, with Prof. Zi-Feng Yang (Guangzhou Institute of Respiratory Health) serving as the corresponding author. The research was funded by China’s National Key Research and Development Program, Guangdong Province’s science research projects, and Guangzhou Medical University’s clinical research initiatives.
References
This study was originally published in the Chinese Medical Journal (2020; 133: 1385–1386) by Wen-Da Guan et al. Guidelines from the National Health Commission of the People’s Republic of China (Trial Version 6, 2020) were cited to support diagnostic criteria.
doi.org/10.1097/CM9.0000000000000792
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