Detecting and Quantifying Multiple Proteins in Clinical Samples in High-Throughput Using Antibody Microarrays

We have developed a high-throughput, semiautomated, quantitative fluorescence-based PCR assay to detect and type herpes simplex virus (HSV) DNA in clinical samples. The detection assay, which uses primers to the type-common region of HSV glycoprotein B (gB), was linear from <10 to 108 copies of HSV DNA/20 μl of sample. Among duplicate samples in reproducibility runs, the assay showed less than 5% variability. We compared the fluorescence-based PCR assay with culture and gel-based liquid hybridization system with 335 genital tract specimens from HSV type 2 (HSV-2)-seropositive persons attending a research clinic and 380 consecutive cerebrospinal fluid (CSF) samples submitted to a diagnostic virology laboratory. Among the 162 culture-positive genital tract specimens, TaqMan PCR was positive for 157 (97%) specimens, whereas the quantitative-competitive PCR was positive for 144 (89%) specimens. Comparisons of the mean titer of HSV DNA detected by the two assays revealed that the mean titer detected by the gel-based system was slightly higher (median, 1 log). These differences in titers were in part related to the fivefold difference in the amount of HSV DNA used in the amplicon standards with the two assays. Among the 380 CSF samples, 42 were positive by both assays, 13 were positive only by the assay with the agarose gel, and 3 were positive only by the assay with the fluorescent probe. To define the subtype of HSV DNA detected in the screening assay, we also designed one set of primers which amplifies the gG regions of both types of HSV and probes which are specific to either HSV-1 (gG1) or HSV-2 (gG2). These probes were labeled with different fluorescent dyes (6-carboxyfluorescein for gG2 and 6-hexachlorofluorescein for gG1) to enable detection in a single PCR. In mixing experiments the probes discriminated the correct subtype in mixtures with up to a 7-log-higher concentration of the opposite subtype. The PCR typing results showed 100% concordance with the results obtained by assays with monoclonal antibodies against HSV-1 or HSV-2. Thus, while the real-time PCR is slightly less sensitive than the gel-based liquid hybridization system, the high throughput, the lack of contamination during processing, the better reproducibility, and the better ability to type the isolates rapidly make the real-time PCR a valuable tool for clinical investigation and diagnosis of HSV infection.

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Validation of a real-time PCR assay for high-throughput detection of Avibacterium paragallinarum in chicken respiratory sites

Journal of Veterinary Diagnostic Investigation ◽

10.1177/1040638719866484 ◽

2019 ◽

Vol 31 (5) ◽

pp. 714-718 ◽

Cited By ~ 2

Author(s):

Kristin A. Clothier ◽

Simone Stoute ◽

Andrea Torain ◽

Beate Crossley

Keyword(s):

Real Time ◽

High Throughput ◽

Real Time Pcr ◽

Limit Of Detection ◽

Bacterial Flora ◽

Clinical Samples ◽

Single Tube ◽

Avibacterium Paragallinarum ◽

Normal Bacterial Flora ◽

Close Relatives

Avibacterium paragallinarum is the causative agent of infectious coryza, a highly contagious respiratory disease in chickens. Given its fastidious nature, this bacterium is difficult to recover and identify, particularly from locations colonized by normal bacterial flora. Standard PCR methods have been utilized for detection but are labor-intensive and not feasible for high-throughput testing. We evaluated a real-time PCR (rtPCR) method targeting the HPG-2 region of A. paragallinarum, and validated a high-throughput extraction for this assay. Using single-tube extraction, the rtPCR detected 4 A. paragallinarum (ATCC 29545T and 3 clinical) isolates with a limit of detection (LOD) of 10 cfu/mL and a PCR efficiency of 89–111%. Cross-reaction was not detected with 33 non– A. paragallinarum, all close relatives from the family Pasteurellaceae. Real-time PCR testing on extracts of 66 clinical samples (choana, sinus, or trachea) yielded 98.2% (35 of 36 on positives, 30 of 30 on negatives) agreement with conventional PCR. Duplicate samples tested in a 96-well format extraction in parallel with the single-tube method produced equivalent LOD on all A. paragallinarum isolates, and 96.8% agreement on 93 additional clinical samples extracted with both procedures. This A. paragallinarum rtPCR can be utilized for outbreak investigations and routine monitoring of susceptible flocks.

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A cell-free DNA metagenomic sequencing assay that integrates the damage response to infection

10.1101/648592 ◽

2019 ◽

Author(s):

Alexandre Pellan Cheng ◽

Philip Burnham ◽

John Richard Lee ◽

Matthew Pellan Cheng ◽

Manikkam Suthanthiran ◽

...

Keyword(s):

Urinary Tract ◽

High Throughput ◽

High Throughput Sequencing ◽

Viral Infections ◽

Clinical Samples ◽

Metagenomic Sequencing ◽

Kidney Transplant Recipients ◽

Bladder Tissue ◽

Cell Free Dna ◽

Free Dna

ABSTRACTHigh-throughput metagenomic sequencing offers an unbiased approach to identify pathogens in clinical samples. Conventional metagenomic sequencing however does not integrate information about the host, which is often critical to distinguish infection from infectious disease, and to assess the severity of disease. Here, we explore the utility of high-throughput sequencing of cell-free DNA after bisulfite conversion to map the tissue and cell types of origin of host-derived cell-free DNA, and to profile the bacterial and viral metagenome. We applied this assay to 51 urinary cfDNA isolates collected from a cohort of kidney transplant recipients with and without bacterial and viral infection of the urinary tract. We find that the cell and tissue types of origin of urinary cell-free DNA can be derived from its genome-wide profile of methylation marks, and strongly depend on infection status. We find evidence of kidney and bladder tissue damage due to viral and bacterial infection, respectively, and of the recruitment of neutrophils to the urinary tract during infection. Through direct comparison to conventional metagenomic sequencing as well as clinical tests of infection, we find this assay accurately captures the bacterial and viral composition of the sample. The assay presented here is straightforward to implement, offers a systems view into bacterial and viral infections of the urinary tract, and can find future use as a tool for the differential diagnosis of infections.

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High throughput detection and genetic epidemiology of SARS-CoV-2 using COVIDSeq next-generation sequencing

PLoS ONE ◽

10.1371/journal.pone.0247115 ◽

2021 ◽

Vol 16 (2) ◽

pp. e0247115

Author(s):

Rahul C. Bhoyar ◽

Abhinav Jain ◽

Paras Sehgal ◽

Mohit Kumar Divakar ◽

Disha Sharma ◽

...

Keyword(s):

High Throughput ◽

Genetic Epidemiology ◽

Confirmatory Test ◽

Clinical Samples ◽

Rt Pcr ◽

Additional Advantage ◽

Depth Analysis ◽

High Concordance ◽

High Throughput Detection ◽

First Time

The rapid emergence of coronavirus disease 2019 (COVID-19) as a global pandemic affecting millions of individuals globally has necessitated sensitive and high-throughput approaches for the diagnosis, surveillance, and determining the genetic epidemiology of SARS-CoV-2. In the present study, we used the COVIDSeq protocol, which involves multiplex-PCR, barcoding, and sequencing of samples for high-throughput detection and deciphering the genetic epidemiology of SARS-CoV-2. We used the approach on 752 clinical samples in duplicates, amounting to a total of 1536 samples which could be sequenced on a single S4 sequencing flow cell on NovaSeq 6000. Our analysis suggests a high concordance between technical duplicates and a high concordance of detection of SARS-CoV-2 between the COVIDSeq as well as RT-PCR approaches. An in-depth analysis revealed a total of six samples in which COVIDSeq detected SARS-CoV-2 in high confidence which were negative in RT-PCR. Additionally, the assay could detect SARS-CoV-2 in 21 samples and 16 samples which were classified inconclusive and pan-sarbeco positive respectively suggesting that COVIDSeq could be used as a confirmatory test. The sequencing approach also enabled insights into the evolution and genetic epidemiology of the SARS-CoV-2 samples. The samples were classified into a total of 3 clades. This study reports two lineages B.1.112 and B.1.99 for the first time in India. This study also revealed 1,143 unique single nucleotide variants and added a total of 73 novel variants identified for the first time. To the best of our knowledge, this is the first report of the COVIDSeq approach for detection and genetic epidemiology of SARS-CoV-2. Our analysis suggests that COVIDSeq could be a potential high sensitivity assay for the detection of SARS-CoV-2, with an additional advantage of enabling the genetic epidemiology of SARS-CoV-2.

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Algorithmic Approach to High-Throughput Molecular Screening for Alpha Interferon-Resistant Genotypes in Hepatitis C Patients

Journal of Clinical Microbiology ◽

10.1128/jcm.36.7.1895-1901.1998 ◽

1998 ◽

Vol 36 (7) ◽

pp. 1895-1901 ◽

Cited By ~ 8

Author(s):

Srinand Sreevatsan ◽

Jack B. Bookout ◽

Fidel M. Ringpis ◽

Mridula R. Pottathil ◽

David J. Marshall ◽

...

Keyword(s):

Hepatitis C ◽

Dna Sequencing ◽

High Throughput ◽

Incubation Temperature ◽

Alpha Interferon ◽

Clinical Samples ◽

Nucleotide Sequence Analysis ◽

Rt Pcr ◽

Hcv Genotypes ◽

Genotype Frequencies

This study was designed to analyze the feasibility and validity of using Cleavase Fragment Length Polymorphism (CFLP) analysis as an alternative to DNA sequencing for high-throughput screening of hepatitis C virus (HCV) genotypes in a high-volume molecular pathology laboratory setting. By using a 244-bp amplicon from the 5′ untranslated region of the HCV genome, 61 clinical samples received for HCV reverse transcription-PCR (RT-PCR) were genotyped by this method. The genotype frequencies assigned by the CFLP method were 44.3% for type 1a, 26.2% for 1b, 13.1% for type 2b, and 5% type 3a. The results obtained by nucleotide sequence analysis provided 100% concordance with those obtained by CFLP analysis at the major genotype level, with resolvable differences as to subtype designations for five samples. CFLP analysis-derived HCV genotype frequencies also concurred with the national estimates (N. N. Zein et al., Ann. Intern. Med. 125:634–639, 1996). Reanalysis of 42 of these samples in parallel in a different research laboratory reproduced the CFLP fingerprints for 100% of the samples. Similarly, the major subtype designations for 19 samples subjected to different incubation temperature-time conditions were also 100% reproducible. Comparative cost analysis for genotyping of HCV by line probe assay, CFLP analysis, and automated DNA sequencing indicated that the average cost per amplicon was lowest for CFLP analysis, at $20 (direct costs). On the basis of these findings we propose that CFLP analysis is a robust, sensitive, specific, and an economical method for large-scale screening of HCV-infected patients for alpha interferon-resistant HCV genotypes. The paper describes an algorithm that uses as a reflex test the RT-PCR-based qualitative screening of samples for HCV detection and also addresses genotypes that are ambiguous.

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Multiplex and On-site PCR detection of swine diseases based on the microfluidic chip system

10.21203/rs.2.21078/v3 ◽

2020 ◽

Author(s):

Yan Jiang ◽

Shan Jiang ◽

Yue Wu ◽

Bin Zhou ◽

Kaiming Wang ◽

...

Keyword(s):

High Throughput ◽

Microfluidic Chip ◽

Detection System ◽

Sampling Site ◽

High Sensitivity ◽

Pcr Detection ◽

Clinical Samples ◽

Pathogenic Microorganisms ◽

Real Time Quantitative Pcr ◽

Inspection And Quarantine

Abstract Background： At present, the process of inspection and quarantine starts with sampling at the customs port, continues with transporting the samples to the central laboratory for inspection experiments, and ends with the inspected results being fed back to the port. This process had the risks of degradation of biological samples and generation of pathogenic microorganisms and did not meet the rapid on-site detection demand because it took a rather long time. Therefore, it is urgently needed to develop a rapid and high-throughput detection assay of pathogenic microorganisms at the customs port. The aim of this study was to develop a microfluidic chip to rapidly detect swine pathogenic microorganisms with high-throughput and higher accuracy. Moreover, this chip will decrease the risk of spreading infection during transportation.Results: A series of experiments were performed to establish a microfluidic chip. The resulting data showed that the positive nucleic acid of four swine viruses were detected by using a portable and rapid microfluidic PCR system, which could achieve a on-site real-time quantitative PCR detection. Furthermore, the detection results of eight clinical samples were obtained within an hour. The detection limit of this microfluidic PCR detection system was as low as 1 copies/μL. The results showed that the high sensitivity and specificity of this chip system in disease detection played an important role in customs inspection and quarantine during customs clearance.Conclusion: The microfluidic PCR detection system established in this study could meet the requirement for rapid detection of samples at the customs port. This chip could avoid the risky process of transporting the samples from the sampling site to the testing lab, and drastically reduce the inspection cycle. Moreover, it would enable parallel inspections on one chip, which greatly raised the efficiency of inspection.

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High-Throughput Mass Cytometry Staining for Immunophenotyping Clinical Samples

STAR Protocols ◽

10.1016/j.xpro.2020.100055 ◽

2020 ◽

Vol 1 (2) ◽

pp. 100055

Author(s):

Emily M. Thrash ◽

Katja Kleinsteuber ◽

Emma S. Hathaway ◽

Matthew Nazzaro ◽

Eric Haas ◽

...

Keyword(s):

High Throughput ◽

Clinical Samples ◽

Mass Cytometry

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High-throughput, Automated Extraction of DNA and RNA from Clinical Samples using TruTip Technology on Common Liquid Handling Robots

Journal of Visualized Experiments ◽

10.3791/50356 ◽

2013 ◽

Author(s):

Rebecca C. Holmberg ◽

Alissa Gindlesperger ◽

Tinsley Stokes ◽

Dane Brady ◽

Nitu Thakore ◽

...

Keyword(s):

High Throughput ◽

Clinical Samples ◽

Automated Extraction ◽

Liquid Handling ◽

Dna And Rna

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Comparison of SARS-CoV-2 RT-PCR on a high-throughput molecular diagnostic platform and the cobas SARS-CoV-2 test for the diagnostic of COVID-19 on various clinical samples

Pathogens and Disease ◽

10.1093/femspd/ftaa061 ◽

2020 ◽

Vol 78 (8) ◽

Cited By ~ 1

Author(s):

Onya Opota ◽

René Brouillet ◽

Gilbert Greub ◽

Katia Jaton

Keyword(s):

High Throughput ◽

Molecular Diagnostic ◽

Clinical Samples ◽

Nasopharyngeal Swab ◽

Test Results ◽

Rt Pcr ◽

Clinical Specimens ◽

Rapid Spread ◽

Bronchial Aspirate ◽

Very High

ABSTRACT Objectives:In order to cope with the rapid spread of the COVID-19 pandemic, we introduced on our in-house high-throughput molecular diagnostic platform (MDx Platform) a real-time reverse transcriptase PCR (RT-PCR) to detect the SARS-CoV-2 from any clinical specimens. The aim of this study was to compare the RT-PCR results obtain with the MDx Platform and the commercial assay cobas SARS-CoV-2 (Roche) on nasopharyngeal swab and other clinical specimens including sputum, bronchial aspirate, bronchoalveolar lavage and anal swabs. Methods: Samples received in our laboratory from patients suspected of COVID-19 (n = 262) were tested in parallel with our MDx platform SARS-CoV-2 PCR and with the cobas SARS-CoV-2 test. Results: The overall agreement between the two tests for all samples tested was 99.24% (260/262), which corresponded to agreements of 100% (178/178) on nasopharyngeal swabs, 95.45% (42/44) on lower respiratory tract specimen with discordant resultS obtained for very high cycle threshold (Ct) value and 100% (40/40) on anorectal swabs. The Ct values for nasopharyngeal swabs displayed an excellent correlation (R2 > 96%) between both tests. Conclusions: The high agreements between the cobas SARS-CoV-2 test and the MDx platform supports the use of both methods for the diagnostic of COVID-19 on various clinical samples. Very few discrepant results may occur at very low viral load.

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