Retro Biotech | Contact

Evaluation of direct-to-PCR (D2P) method for molecular diagnosis of infectious diseases

Abstract: This study evaluates the performance of the Direct-to-PCR (D2P) method as a streamlined, extractionindependent alternative to conventional nucleic acid extraction techniques for diagnosing urinary tract infections, sexually transmitted infections, and respiratory tract infections. The D2P approach employs proprietary antimicrobial peptide-based lysis buffers tailored for bacterial, fungal, and viral targets, enabling direct amplification from clinical and contrived specimens without column- or bead-based purification. Comparative analyses were conducted against silica column-based (QIAGEN) and magnetic bead-based (KingFisher) extraction methods using both microbial reference isolates and 116 residual clinical samples. Results demonstrate that the D2P method yields comparable sensitivity and specificity to conventional extraction workflows across a diverse panel of pathogens—including Gram-negative and Gram-positive bacteria, Candida species, ssRNA viruses (e.g., CoV-229E, Parainfluenza Virus 1 and 2), and dsDNA viruses (e.g., HSV, HAdV). Notably, D2P outperformed both QIAGEN and KingFisher in extracting nucleic acids from Candida auris, a multidrug-resistant fungal pathogen. Limit of detection and amplification efficiency remained within acceptable ranges across all platforms, with R2 values between 0.92 and 0.99, and slopes consistent with MIQE standards. The D2P protocol reduced total sample processing time from ~120 min to ~45 min, minimized hands-on steps, and demonstrated effective performance in turbid or hemolyzed samples—making it suitable for high-throughput and resource-limited settings. However, limitations were observed in samples with high PCR-inhibitor content or low target yield, and broader validation across additional matrices is recommended. These findings support D2P as a reliable, efficient, and scalable molecular diagnostic alternative with broad clinical utility. Integration of D2P into diagnostic workflows could enhance access to rapid, cost-effective pathogen detection in both centralized laboratories and decentralized or point-of-care environments.

Streamlining fungal diagnostics: Evaluation of a Direct-to-PCR extraction-free workflow for candida detection

Abstract: Fungal infections are an escalating health threat, and delays from conventional nucleic acid extraction hinder timely diagnosis and treatment. This study evaluated the diagnostic performance of a novel extraction-free technology, Direct-to-PCR (D2P; Scienetix, Tyler, TX, USA), for the detection of clinically significant Candida species (C. albicans, C. glabrata, C. auris, C. parapsilosis, and C. tropicalis). D2P was compared against conventional silica column-based (Qiagen) and magnetic bead-based (KingFisher) extraction methods, using microbial reference isolates, residual clinical specimens, and limit-of-detection analyses. Diagnostic sensitivity and specificity of D2P were comparable to conventional approaches, with specificity ranging from 96.77 % to 100 %. Concordance between methods was high, with Cohen’s kappa coefficients (κ=0.93–1.00). Limit-of-detection analyses demonstrated strong analytical sensitivity, with excellent linearity (R²=0.924–0.999) and low replicate variability (coefficient of variation 0.2–6.3 %). Statistical comparison of cycle threshold values revealed no significant differences between methods (p > 0.05), supporting equivalent nucleic acid recovery without the need for time-intensive extraction steps. Despite these strengths, the study has limitations. The relatively small number of residual clinical specimens (n = 40) may restrict the generalizability of findings, and further validation across broader patient populations, specimen types, and clinical settings is warranted. Nevertheless, D2P offers a streamlined, rapid diagnostic workflow that reduces turnaround times and enhances accessibility, particularly in resource-limited environments. Wider adoption of extraction-free PCR platforms such as D2P could facilitate earlier detection of invasive candidiasis, improve clinical outcomes, and mitigate healthcare-associated morbidity and mortality attributable to fungal infections.

Dataset for comparative analysis of precision metagenomics and traditional methods in urinary tract infection diagnostics

Abstract: This study presents a comprehensive dataset comparing three diagnostic methodologies—microbial culture, polymerase chain reaction (PCR), and precision metagenomics (precision metagenomics)—for the detection and classification of uropathogens in urine samples from patients with suspected urinary tract infections (UTIs). While microbial culture remains the gold standard for UTI diagnosis, it has limitations in sensitivity, particularly for fastidious or nonculturable microorganisms. PCR offers higher sensitivity but is restricted to pre-targeted organisms, limiting its diagnostic range. Precision Metagenomics, a target-agnostic sequencing method, provides a more inclusive approach by enabling the identification of a broad spectrum of pathogens, including bacteria, viruses, fungi, and parasites, without prior knowledge of the organisms. The dataset includes 47 urine samples, each analyzed by microbial culture, PCR, and precision metagenomics, followed by bioinformatic classification using the Explify® platform. precision metagenomics identified significantly more uropathogens (62 distinct organisms) compared to PCR (19 organisms) and microbial culture (13 organisms), with 98 % of samples testing positive for polymicrobial infections via precision metagenomics. The precision metagenomics method demonstrated superior diagnostic yield by detecting pathogens that were missed by both microbial culture and PCR, particularly in culture-negative and PCRnegative cases. This dataset holds substantial reuse potential for further research into the microbiome of urinary tract infections, pathogen discovery, antimicrobial resistance studies, and the development of more accurate diagnostic models for UTI management. By offering insights into both polymicrobial infections and rare pathogens, this dataset supports the advancement of diagnostic strategies for complex and chronic UTIs.

Advantage of precision metagenomics for urinary tract infection diagnostics

History shows that usual care testing is not the diagnostic solution for recurrent and complex UTI. This study supports that PM offers prospects to bridge the UTI diagnostic gap. This approach allows a workflow where laboratorians can qualify, quantify, and phenotypically classify pathogenicity more readily through bioinformatic platforms like Explify®, essentially providing dissected results across a broad array of input types and quantities for timely and accurate empiric UTI treatment. Moreover, PM offers potential for building effective diagnostic models beyond usual care testing in complex and coinfected UTI diseases. Future studies should assess the impact of PM-guided UTI management on clinical outcomes.

SARS-CoV-2 Next Generation Sequencing (NGS) data from clinical isolates from the East Texas Region of the United States

Conclusion: The SARS-CoV-2 virus has evolved throughout the pandemic and is likely to continue evolving into new variants. Some of these variants may affect functional properties, including infectivity, interactions with host immunity, and disease severity. And compromised vaccine efficacy is an emerging concern with every new viral variant. Next-generation sequencing (NGS) has emerged as the tool of choice for discovering new variants and understanding the transmission dynamics of SARS-CoV-2. Deciphering the SARS-CoV-2 genome has enabled epidemiological survivance and forecast of altered etiologically. Clinical presentations of the infection are influenced by comorbidities such as age, immune status, diabetes, and the infecting variant. Thus, clinical management and vaccine efficacy may differ for new variants. For example, some monoclonal antibody treatments are variantspecific, and some vaccines are less efficacious against the omicron and delta variants of SARS-CoV-2. Consequently, determining the local outbreaks and monitoring SARS-CoV-2 Variants of Concern (VOC) is one of the primary strategies for the pandemic’s containment. Although next-generation sequencing (NGS) is a gold standard for genomic surveillance and variant discovery, the assays are not approved for variant diagnosis for clinical decision-making. Advanta Genetics, Texas, USA, optimized Illumina COVID-seq protocol to reduce cost without compromising accuracy and validated the Illumina COVID-Seq assay as a Laboratory Developed Test (LDT) according to the guidelines prescribed by the College of American Pathologists (CAP) and Clinical Laboratory Improvement Amendments (CLIA). The whole genome of the virus was sequenced in (n = 161) samples from the East Texas region using the Illumina MiniSeq® instrument and analyzed by using Illumina baseSpace (https://basespace.illumina.com) bioinformatics pipeline. Briefly, the library was prepared by using Illumina COVIDSeq research use only (RUO) kit, and the individual libraries were normalized using the DNA concentration measured by Qubit Flex Fluorometer, and the pooled libraries were sequenced on Illumina MiniSeq® Instrument. Illumina baseSpace application was used for sequencing QC, FASTQ generation, genome assembly, and identification of SARS-CoV-2 variants. This whole genome shotgun project (n = 161) has been deposited at GISAID.

Optimization of the Illumina COVIDSeq™ protocol for decentralized, cost-effective genomic surveillance

Conclusion: COVID incident rate is sloping downward globally so is the financial support for high throughput genomic surveillance. However, persistent surveillance of circulating genomic variants in the community is crucial for scrutinizing the transmission dynamics of existing variants and tracking the emergence of new variants. We have optimized the SARS-CoV-2 variant detection assays for sequencing small batches (n = 48) of samples on a low-cost instrument (Illumina MiniSeq) which is ideal for small laboratories often not supported by centralized funding sources. Furthermore, surveillance data from such laboratories is critical for broadening the representation of the under-served population in global databases such as GSAID. Simultaneous sequencing of ~3000 samples using COVIDSeq™ EUA kit on Illumina NovaSeq (Price~$1.0 million + $100,000/ year maintenance cost) instrument is the most cost-effective (~$21.20/sample) option for SARS-CoV-2 genome sequencing often applied for mass surveillance. However, this published low cost is unattainable for independent clinical laboratories because of high capital investment and large batch size. Standard COVIDSeq™ protocol in Illumina’s EUA test allows the sequencing of up to 384 samples on the NextSeq 550 at a lower cost ($25.33/sample), which is slightly lower than the estimated cost ($43.27/sample) of this modified protocol for MinSeq. However, cost-effective testing will still require pooling of >300 samples to achieve ~$25/sample reagent cost. Capital investment for NextSeq 550 instrument (Cost ~$300,000 + ~$30,000/year maintenance cost) is still significantly higher than MiniSeq (Cost ~$50,000 + ~$5,000/year maintenance cost). Pre-pooling normalization has reduced the library preparation and sequencing cost on MiniSeq instruments close to the NextSeq or NovaSeq. Therefore, the modified protocol could empower small resource-limited laboratories to contribute to local genomic surveillance. We have adopted this modified protocol for sequencing 153 genomes from East Texas, USA, and compared the results with PCR-based variant detection [8]. High accuracy and reproducibility of this approach have been demonstrated in validating the COVIDSeq™ RUO assay for clinical application according to Clinical Laboratory Improvement Amendments (CLIA) and College of American Pathologists (CAP) guidelines [6]. We have only scrutinized this procedure for accurate detection of circulating variants rather than detecting new mutations and variant frequency in the mixed variant population. Secondly, the cost estimates presented in this study are for the core reagents (library preparation and sequencing) only. Personnel cost is a significant expense in processing NGS samples, which can vary significantly according to geographical location. For example, capital investment and the cost of imported reagents are often the limiting factors in low-income countries, not so much the trained personnel. Therefore, this cost-effective approach can still benefit the low-throughput sequencing for monitoring emerging variants of SARS-CoV-2 and support decentralized genomic surveillance, particularly in resource-limited settings.

Deciphering Microbiota of Acute Upper Respiratory Infections: A Comparative Analysis of PCR and mNGS Methods for Lower Respiratory Trafficking Potential

Conclusions: Much of respiratory medicine is reliant on timely and precise diagnostics for critical treatment. However, respiratory infections remain a leading cause of global mortality and morbidity despite advances in diagnosis and treatment. In this study, we (a) compared nasopharyngeal samples from patients suspected of acute upper respiratory infection between a commercially available PCR assay and a targeted hybridization-capture-based mNGS workflow and (b) demonstrated that the hybridized approach may provide tremendous advantage in deciphering the etiological agent of respiratory (co)infections and provide clinical relevance for trafficking potential. This is important because the trafficking potential from the upper to the lower respiratory tract and infection severity depend on pathogen virulence, concomitant infections, and underlying respiratory comorbidities [81]. This is significant to respiratory medicine because this technology can be used to supplement current syndromic-based tests, and data can quickly and effectively be phenotypically characterized for clinical (co)infection and comorbid consideration. This has significance for laboratory medicine because it demonstrated that this approach can rapidly be interpreted with a user-friendly and reliable platform for collective intention without overburdening laboratory investments in technology and people [82,83]. Furthermore, this approach could be advanced into pan-microbial diagnostic testing that utilizes a single workflow for all specimen types. Although we have demonstrated the analytical advantage of a targeted hybridization-capture-based mNGS workflow over targeted PCR analysis, further investigations are required to establish the clinical relevance of phenotypic classifications and their value to trafficking predispositions and utility in respiratory medicine.

COVIDSeq as Laboratory Developed Test (LDT) for Diagnosis of SARS-CoV-2 Variants of Concern (VOC)

Conclusions: Rapid classification and detection of SARS-CoV-2 variants have been critical in comprehending the virus's transmission dynamics. Clinical manifestation of the infection is influenced by comorbidities such as age, immune status, diabetes, and the infecting variant. Thus, clinical management may differ for new variants. For example, some monoclonal antibody treatments are variant-specific. Yet, a U.S. Food and Drug Administration (FDA)-approved test for detecting the SARS-CoV-2 variant is unavailable. A laboratory-developed test (LDT) remains a viable option for reporting the infecting variant for clinical intervention or epidemiological purposes. Accordingly, we have validated the Illumina COVIDSeq assay as an LDT according to the guidelines prescribed by the College of American Pathologists (CAP) and Clinical Laboratory Improvement Amendments (CLIA). The limit of detection (LOD) of this test is Ct<30 (~15 viral copies) and >200X genomic coverage, and the test is 100% specific in the detection of existing variants. The test demonstrated 100% precision in inter-day, intra-day, and intralaboratory reproducibility studies. It is also 100% accurate, defined by reference strain testing and split sample testing with other CLIA laboratories. Advanta Genetics LDT COVIDSeq has been reviewed by CAP inspectors and is under review by FDA for Emergency Use Authorization.

Confirming Multiplex RT-qPCR Use in COVID-19 with Next-Generation Sequencing: Strategies for Epidemiological Advantage

Conclusion: There are two important takeaways from this study. First, the NGS data provided further evidence of the rapid evolution of SARS-CoV-2 lineages including the highly transmissible Delta variant in the East Texas region and suggests the continued threat of COVID-19. This finding is consistent with other research and further supports the need for rapid, cost-effective monitoring of variant mutations. Second, the current study endorses the potential of RT-qPCR assays as a solution for more accessible variant monitoring. )e data showed concordance with RT-qPCR and NGS analysis for specific SARS-CoV-2 lineages and characteristic mutations. Thus, the deployment of RT-qPCR testing for the detection of known SARS-CoV-2 variants may be extremely beneficial. The key difference between the NGS and RT-qPCR is discovery power, scalability, and throughput. Both technologies are reliable and highly sensitive. RT-qPCR can detect only known sequences with help of specific probes and primers. In contrast, NGS does not need prior information about the sequence, but NGS is less cost-effective for low target numbers and is a time-consuming method. NGS can detect thousands of targeted regions with single-base resolution. RT-qPCR is cost-effective, and its familiar workflow made the detection of a limited set of variants and low target numbers easy [34]. Accordingly, is it suggested that RT-qPCR is a quick and cost-effective alternative to sequencing for screening known mutations of SARS-CoV-2 for clinical and epidemiological interest, especially in developing countries where COVID-19 diagnostic centers are limited by regional sequencing laboratories for screening the mutations in the SARS-CoV-2 clinical samples. )e findings in this study depict great potential for RT-qPCR to be an effective strategy offering several epidemiological advantages.

Publications