In brief, Multiplex RT-PCR is generally better for routine diagnosis of known gut pathogens because it is faster, more sensitive and specific, and more cost-effective.
Reverse transcription polymerase chain reaction (RT-PCR) and shotgun metagenomic testing have distinct strengths for identifying gut pathogens. RT-PCR-based multiplex panels demonstrate higher sensitivity and specificity (96-97% and 99-100%, respectively) for detecting known pathogens, with rapid turnaround times of 2-5 hours.[1-3] In contrast, shotgun metagenomics shows lower sensitivity (86-91% for bacterial pathogens) but offers the unique advantage of detecting novel, unexpected, or difficult-to-culture organisms without requiring prior knowledge of the pathogen.[4-5]
Multiplex RT-PCR panels consistently achieve higher diagnostic yields than conventional methods, detecting pathogens in 28-40% of samples versus 15-20% with traditional culture-based approaches.[1-2] These panels excel at identifying viral pathogens, mixed infections (detected in 11-35% of positive samples), and provide results that enable earlier antibiotic stewardship decisions.[1][3] However, RT-PCR cannot provide antimicrobial susceptibility data or subtyping information critical for outbreak surveillance.[2]
Shotgun metagenomics offers complementary capabilities by providing species and strain-level identification, functional genomic information, and detection of antimicrobial resistance genes.[5-6] Metatranscriptomics (RNA-based shotgun sequencing) demonstrates superior sensitivity compared to DNA-based metagenomics for many pathogens and can distinguish active infection from colonization through RNA/DNA ratios.[6] The main limitations include higher cost (approximately 9-fold more expensive than conventional methods), longer turnaround times, computational complexity, and challenges with background microbiome interference.[2][4-5]
For routine clinical diagnosis of known gut pathogens, multiplex RT-PCR panels are currently superior due to their speed, sensitivity, and cost-effectiveness. Shotgun metagenomics serves best as a complementary tool for cases where RT-PCR is negative despite high clinical suspicion, for outbreak investigation requiring strain typing, or when novel pathogens are suspected.[4-5][7]
- Comparison of the Luminex® NxTAG® Gastrointestinal Pathogen Panel to Traditional Diagnostic Methods for Detecting Diarrhoea-Associated Gastroenteritis. Journal of Medical Microbiology. 2025. Wilson K, Beckett P, Collins M.
- Comparative Evaluation of the Detection Rate, Workflow and Associated Costs of a Multiplex PCR Panel Versus Conventional Methods in Diagnosis of Infectious Gastroenteritis. Journal of Medical Microbiology. 2024. Ambrosius-Eichner J, Hogardt M, Berger A, et al.
- PCR-Based Versus Conventional Stool Testing in Hospitalized Patients With Diarrhea: Diagnostic Yield, Clinical Impact, and Stewardship Implications. Microorganisms. 2025. Boeriu A, Andone A, Dobru D, et al.
- Evaluation of Shotgun Metagenomics as a Diagnostic Tool for Infectious Gastroenteritis. PloS One. 2025. Haugum K, Ravi A, Afset JE, Ås CG.
- Clinical Metagenomics Is Increasingly Accurate and Affordable to Detect Enteric Bacterial Pathogens in Stool. Microorganisms. 2022. Peterson CL, Alexander D, Chen JC, et al.
- Enhancing Infectious Intestinal Disease Diagnosis Through Metagenomic and Metatranscriptomic Sequencing of 1000 Human Diarrhoeal Samples. Genome Medicine. 2025. Cunningham-Oakes E, Perez-Sepulveda BM, Li Y, et al.
- Shotgun Metagenomics for the Diagnosis of Infections: A Prospective Study. The Journal of Infection. 2025. Surgers L, Lafont C, Lamoureux C, et al.