Development of Two Multiplex Real-Time PCR Assays for the Rapid Detection of RNA and DNA Viruses Associated with Gastroenteritis in Pediatric Patients

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected millions within just a few months, causing severe respiratory disease and mortality. Assays to monitor SARS-CoV-2 growth in vitro depend on time-consuming and costly RNA extraction steps, hampering progress in basic research and drug development efforts. Here, we developed a simplified quantitative real-time PCR assay that bypasses viral RNA extraction steps and can monitor SARS-CoV-2 growth from a small amount of cell culture supernatants. In addition, we show that this approach is easily adaptable to numerous other RNA and DNA viruses. Using this assay, we screened the activities of a number of compounds that were predicted to alter SARS-CoV-2 entry and replication as well as HIV-1-specific drugs in a proof-of-concept study. We found that E64D (inhibitor of endosomal proteases cathepsin B and L) and apilimod (endosomal trafficking inhibitor) potently decreased the amount of SARS-CoV-2 RNA in cell culture supernatants with minimal cytotoxicity. Surprisingly, we found that the macropinocytosis inhibitor ethylisopropylamiloride (EIPA) similarly decreased SARS-CoV-2 RNA levels in supernatants, suggesting that entry may additionally be mediated by an alternative pathway. HIV-1-specific inhibitors nevirapine (a nonnucleoside reverse transcriptase inhibitor [NNRTI]), amprenavir (a protease inhibitor), and allosteric integrase inhibitor 2 (ALLINI-2) modestly inhibited SARS-CoV-2 replication, albeit the 50% inhibitory concentration (IC50) values were much higher than that required for HIV-1. Taking the data together, this simplified assay will expedite basic SARS-CoV-2 research, be amenable to mid-throughput screening assays (i.e., drug, CRISPR, small interfering RNA [siRNA], etc.), and be applicable to a broad number of RNA and DNA viruses. IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the coronavirus disease 2019 (COVID-19) pandemic, is continuing to cause immense respiratory disease and social and economic disruptions. Conventional assays that monitor SARS-CoV-2 growth in cell culture rely on costly and time-consuming RNA extraction procedures, hampering progress in basic SARS-CoV-2 research and development of effective therapeutics. Here, we developed a simple quantitative real-time PCR assay to monitor SARS-CoV-2 growth in cell culture supernatants that does not necessitate RNA extraction and that is as accurate and sensitive as existing methods. In a proof-of-concept screen, we found that E64D, apilimod, EIPA, and remdesivir can substantially impede SARS-Cov-2 replication, providing novel insight into viral entry and replication mechanisms. In addition, we show that this approach is easily adaptable to numerous other RNA and DNA viruses. This simplified assay will undoubtedly expedite basic SARS-CoV-2 and virology research and be amenable to use in drug screening platforms to identify therapeutics against SARS-CoV-2.

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Development of Candida-Specific Real-Time PCR Assays for the Detection and Identification of Eight Medically Important Candida Species

Microbiology Insights ◽

10.4137/mbi.s38517 ◽

2016 ◽

Vol 9 ◽

pp. MBI.S38517 ◽

Cited By ~ 15

Author(s):

Jing Zhang ◽

Guo-Chiuan Hung ◽

Kenjiro Nagamine ◽

Bingjie Li ◽

Shien Tsai ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Candida Species ◽

Rapid Detection ◽

Corneal Transplantation ◽

High Sensitivity ◽

Turnaround Time ◽

Pcr Assays ◽

Primer Sets ◽

Pcr Primer

Culture-based identification methods have been the gold standard for the diagnosis of fungal infection. Currently, molecular technologies such as real-time PCR assays with short turnaround time can provide desirable alternatives for the rapid detection of Candida microbes. However, most of the published PCR primer sets are not Candida specific and likely to amplify DNA from common environmental contaminants, such as Aspergillus microbes. In this study, we designed pan- Candida primer sets based on the ribosomal DNA-coding regions conserved within Candida but distinct from those of Aspergillus and Penicillium. We demonstrate that the final two selected pan- Candida primer sets would not amplify Aspergillus DNA and could be used to differentiate eight medically important Candida pathogens in real-time PCR assays based on their melting profiles, with a sensitivity of detection as low as 10 fg of Candida genomic DNA. Moreover, we further evaluated and selected species-specific primer sets covering Candida albicans, Candida glabrata, Candida tropicalis, and Candida dubliniensis and show that they had high sensitivity and specificity. These real-time PCR primer sets could potentially be assembled into a single PCR array for the rapid detection of Candida species in various clinical settings, such as corneal transplantation.

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Real-time PCR assays for rapid detection ofZeugodacus cucumisandBactrocera jarvisi(Diptera: Tephritidae) for quarantine application

Journal of Applied Entomology ◽

10.1111/jen.12577 ◽

2018 ◽

Vol 143 (1-2) ◽

pp. 155-163 ◽

Cited By ~ 1

Author(s):

Dongmei Li ◽

Sreelakshmi Nair ◽

Diane Anderson ◽

Prasad Doddala ◽

Disna N. Gunawardana ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Rapid Detection ◽

Pcr Assays

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Development of Conventional and Real-Time PCR Assays for the Rapid Detection of Group B Streptococci

Clinical Chemistry ◽

10.1093/clinchem/46.3.324 ◽

2000 ◽

Vol 46 (3) ◽

pp. 324-331 ◽

Cited By ~ 106

Author(s):

Danbing Ke ◽

Christian Ménard ◽

François J Picard ◽

Maurice Boissinot ◽

Marc Ouellette ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Rapid Detection ◽

Culture Method ◽

Conventional Pcr ◽

Pcr Assay ◽

Group B Streptococci ◽

Standard Culture ◽

Pcr Assays ◽

Group B

Abstract Background: Group B streptococci (GBS), or Streptococcus agalactiae, are the leading bacterial cause of meningitis and bacterial sepsis in newborns. Currently available rapid methods to detect GBS from clinical specimens are unsuitable for replacement of culture methods, mainly because of their lack of sensitivity. Methods: We have developed a PCR-based assay for the rapid detection of GBS. The cfb gene encoding the Christie-Atkins-Munch-Petersen (CAMP) factor was selected as the genetic target for the assay. The PCR primers were initially tested by a conventional PCR method followed by gel electrophoresis. The assay was then adapted for use with the LightCyclerTM. For this purpose, two fluorogenic adjacent hybridization probes complementary to the GBS-specific amplicon were designed and tested. In addition, a rapid sample-processing protocol was evaluated by colony-forming unit counting and PCR. A total of 15 vaginal samples were tested by both standard culture method and the two PCR assays. Results: The conventional PCR assay was specific because it amplified only GBS DNA among 125 bacterial and fungal species tested, and was able to detect all 162 GBS isolates from various geographical areas. This PCR assay allowed detection of as few as one genome copy of GBS. The real-time PCR assay was comparable to conventional PCR assay in terms of sensitivity and specificity, but it was more rapid, requiring only ∼30 min for amplification and computer-based data analysis. The presence of vaginal specimens had no detrimental effect on the sensitivity of the PCR with the sample preparation protocol used. All four GBS-positive samples identified by the standard culture method were detected by the two PCR assays. Conclusion: These assays provide promising tools for the rapid detection and identification of GBS.

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