Development of a quantitative one-step multiplex RT-qPCR assay for the detection of SARS-CoV-2 in a biological matrix

Abstract BackgroundMosquitoes are the deadliest animals in the world. Their ability to carry and spread diseases to humans causes millions of deaths every year. Due to the lack of efficient vaccines, the control of mosquito-borne diseases often relies on management of the vector. Traditional control methods such as source reduction and chemical insecticides, have proven not to be sufficient to prevent the proliferation and spread of mosquito populations. The sterile insect technique (SIT) is an additional control method that can be combined with other control tactics to suppress specific mosquito populations. The SIT requires the mass-rearing and release of sterile males that would induce sterility in the wild female population. Samples collected from the environment for laboratory colonization, as well as released males, should be free from mosquito-borne viruses (MBV). Therefore, efficient detection methods with defined detection limits for MBV are required. Although a one-step reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) method was developed to detect arboviruses in human and mosquito samples, its detection limit in mosquito samples has yet to be defined. MethodsWe evaluated the detection sensitivity of one step RT-qPCR for targeted arboviruses in large mosquito pools, using pools of non-infected mosquitoes of various sizes (165, 320 and 1600 mosquitoes) containing one infected mosquito body with defined virus titers of chikungunya virus (CHIKV), usutu virus (USUV), West Nile (WNV) virus and Zika virus (ZIKV).ResultsCHIK, USUV, ZIKV, and WNV virus were detected in all tested pools using the RT-qPCR assay. Moreover, in the largest mosquito pools (1600 mosquitoes), RT-qPCR was able to detect the targeted viruses using different total RNA quantities (10, 1, 0.1 ng per reaction) as a template. Correlating the virus titer with the total RNA quantity allowed predicting the maximum number of mosquitoes per pool in which the RT-qPCR can theoretically detect the virus infection. The corresponding equation uses a Ct value of 36 as a cut-off value for virus detection and a virus copy number of 108 for the positive mosquito body. Based on this formula, the detection limits of CHIK, USUV, ZIKV and WNV were 5.08 x 105, 8.74 x 106, 2.33 x 107, and 5.24 x 105, respectively.ConclusionMosquito borne viruses can be reliably detected by RT-qPCR assay in pools of mosquitoes exceeding 1000 specimens. This will represent an important step to expand pathogen-free colonies for mass-rearing sterile males for programmes that have an SIT component.

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An Ultrafast One-Step qRT-PCR Assay for Detection of SARS-CoV-2

10.21203/rs.3.rs-582897/v1 ◽

2021 ◽

Author(s):

Jadranka Milosevic ◽

Mengrou Lu ◽

Wallace Greene ◽

Hong-Zhang He ◽

Si-Yang Zheng

Keyword(s):

Qpcr Assay ◽

Nasopharyngeal Swab ◽

Pcr Assay ◽

Running Time ◽

Clinical Sensitivity ◽

Qrt Pcr ◽

One Step

Abstract We developed an ultrafast one-step RT-qPCR assay for SARS-CoV-2 detection, which can be completed in only 30 mins on benchtop Bio-Rad CFX96. The assay significantly reduces the running time of conventional RT-qPCR: reduced RT step from 10 to 1 min, and reduced PCR cycle of denaturation from 10 to 1 s and extension from 30 to 1 s. A cohort of 60 nasopharyngeal swab samples testing showed that the assay had a clinical sensitivity of 100% and a clinical specificity of 100%.

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40 minutes RT-qPCR Assay for Screening Spike N501Y and HV69-70del Mutations

10.1101/2021.01.26.428302 ◽

2021 ◽

Author(s):

Gulay Korukluoglu ◽

Mustafa Kolukirik ◽

Fatma Bayrakdar ◽

Gozde Girgin Ozgumus ◽

Ayse Basak Altas ◽

...

Keyword(s):

Next Generation Sequencing ◽

Internal Control ◽

Sanger Sequencing ◽

Limit Of Detection ◽

Qpcr Assay ◽

Rapid Screening ◽

Clinical Samples ◽

One Step ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

ABSTRACTA one-step reverse transcription and real-time PCR (RT-qPCR) test was developed for rapid screening (40 minutes) of the Spike N501Y and HV69-70del mutations in SARS-CoV-2 positive samples. The test also targets a conserved region of SARS-CoV-2 Orf1ab as an internal control. The samples containing both the N501Y and HV69-70del mutations are concluded as VOC-202012/01 positive. Samples suspected to be positive for B.1.351 or P.1 are the N501Y positive and HV69-70del negative cases. Limit of detection (LOD) of the kit for Orf1ab target is 500 copies/mL, while that of the N501, Y501 and HV69-70del targets are 5000 copies/mL. The developed assay was applied to 165 clinical samples containing SARS-CoV-2 from 32 different lineages. The SARS-CoV-2 lineages were determined via the next-generation sequencing (NGS). The RT-qPCR results were in 100% agreement with the NGS results that 19 samples were N501Y and HV69-70del positive, 10 samples were N501Y positive and HV69-70del negative, 1 sample was N501Y negative and HV69-70del positive, and 135 samples were N501Y and HV69-70del negative. All the VOC-202012/01 positive samples were detected in people who have traveled from England to Turkey. The RT-qPCR test and the Sanger sequencing was further applied to 1000 SARS-CoV-2 positive clinical samples collected in Jan2021 from the 81 different provinces of Turkey. The RT-qPCR results were in 100% agreement with the Sanger sequencing results that 32 samples were N501Y positive and HV69-70del negative, 4 samples were N501Y negative and HV69-70del positive, 964 samples were N501Y and HV69-70del negative. The specificity of the 40 minutes RT-qPCR assay relative to the sequencing-based technologies is 100%. The developed assay is an advantageous tool for timely and representative estimation of the N501Y positive variants’ prevalence because it allows testing a much higher portion of the SARS-CoV-2 positives in much lower time compared to the sequencing-based technologies.

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Corrigendum to ‘Development of a duplex one-step RT-qPCR assay for the simultaneous detection of Apple scar skin viroidand plant RNA internal control’ [J. Virol. Methods (2015) 100–105]

Journal of Virological Methods ◽

10.1016/j.jviromet.2016.07.027 ◽

2016 ◽

Vol 235 ◽

pp. 196

Author(s):

Subuhi Khan ◽

John Mackay ◽

Lia Liefting ◽

Lisa Ward

Keyword(s):

Internal Control ◽

Simultaneous Detection ◽

Qpcr Assay ◽

One Step ◽

Scar Skin

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One-Step Multiplex RT-qPCR Assay for the Detection of Peste des petits ruminants virus, Capripoxvirus, Pasteurella multocida and Mycoplasma capricolum subspecies (ssp.) capripneumoniae

PLoS ONE ◽

10.1371/journal.pone.0153688 ◽

2016 ◽

Vol 11 (4) ◽

pp. e0153688 ◽

Cited By ~ 15

Author(s):

Tirumala Bharani Kumar Settypalli ◽

Charles Euloge Lamien ◽

Joachim Spergser ◽

Mamadou Lelenta ◽

Abel Wade ◽

...

Keyword(s):

Pasteurella Multocida ◽

Qpcr Assay ◽

Peste Des Petits Ruminants ◽

Mycoplasma Capricolum ◽

One Step

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An Ultrafast One-Step Quantitative Reverse Transcription–Polymerase Chain Reaction Assay for Detection of SARS-CoV-2

Frontiers in Microbiology ◽

10.3389/fmicb.2021.749783 ◽

2021 ◽

Vol 12 ◽

Author(s):

Jadranka Milosevic ◽

Mengrou Lu ◽

Wallace Greene ◽

Hong-Zhang He ◽

Si-Yang Zheng

Keyword(s):

Polymerase Chain Reaction ◽

Reverse Transcription ◽

Polymerase Chain Reaction Assay ◽

Qpcr Assay ◽

Nasopharyngeal Swab ◽

Chain Reaction ◽

Running Time ◽

Clinical Sensitivity ◽

One Step ◽

Polymerase Chain

We developed an ultrafast one-step RT-qPCR assay for SARS-CoV-2 detection, which can be completed in only 30 min on benchtop Bio-Rad CFX96. The assay significantly reduces the running time of conventional RT-qPCR: reduced RT step from 10 to 1 min, and reduced the PCR cycle of denaturation from 10 to 1 s and extension from 30 to 1 s. A cohort of 60 nasopharyngeal swab samples testing showed that the assay had a clinical sensitivity of 100% and a clinical specificity of 100%.

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High sensitivity of one-step real-time reverse transcription quantitative PCR to detect low virus titers in large mosquito pools

10.21203/rs.3.rs-32534/v3 ◽

2020 ◽

Author(s):

Zhaoyang Tang ◽

Hanano Yamada ◽

Carina Kraupa ◽

Sumejja Canic ◽

Núria Busquets ◽

...

Keyword(s):

Quantitative Polymerase Chain Reaction ◽

Virus Titer ◽

Mass Rearing ◽

Qpcr Assay ◽

Detection Sensitivity ◽

Infected Mosquito ◽

Female Population ◽

Total Rna ◽

Quality Control Process ◽

One Step

Abstract BackgroundMosquitoes are the deadliest animals in the world. Their ability to carry and spread diseases to humans causes millions of deaths every year. Due to the lack of efficient vaccines, the control of mosquito-borne diseases primarily relies on the management of the vector. Traditional control methods such as source reduction and chemical insecticides, have proven to be sufficient to prevent the proliferation and spread of mosquito populations. The sterile insect technique (SIT) is an additional control method that can be combined with other control tactics to suppress specific mosquito populations. The SIT requires the mass-rearing and release of sterile males with the aim to induce sterility in the wild female population. Samples collected from the environment for laboratory colonization, as well as the released males, should be free from mosquito-borne viruses (MBV). Therefore, efficient detection methods with defined detection limits for MBV are required. Although a one-step reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) method was developed to detect arboviruses in human and mosquito samples, its detection limit in mosquito samples has yet to be defined.MethodsWe evaluated the detection sensitivity of one step RT-qPCR for targeted arboviruses in large mosquito pools, using pools of non-infected mosquitoes of various sizes (165, 320 and 1600 mosquitoes) containing one infected mosquito body with defined virus titers of chikungunya virus (CHIKV), usutu virus (USUV), West Nile virus (WNV) and Zika virus (ZIKV).ResultsCHIK, USUV, ZIKV, and WNV virus were detected in all tested pools using the RT-qPCR assay. Moreover, in the largest mosquito pools (1600 mosquitoes), RT-qPCR was able to detect the targeted viruses using different total RNA quantities (10, 1, 0.1 ng per reaction) as a template. Correlating the virus titer with the total RNA quantity allowed the prediction of the maximum number of mosquitoes per pool in which the RT-qPCR can theoretically detect the virus infection.ConclusionMosquito borne viruses can be reliably detected by RT-qPCR assay in pools of mosquitoes exceeding 1000 specimens. This will represent an important step to expand pathogen-free colonies for mass-rearing sterile males for programmes that have an SIT component by reducing the time and the manpower needed to conduct this quality control process.

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High sensitivity of one-step real-time reverse transcription quantitative PCR to detect low virus titers in large mosquito pools

10.21203/rs.3.rs-32534/v2 ◽

2020 ◽

Author(s):

Zhaoyang Tang ◽

Hanano Yamada ◽

Carina Kraupa ◽

Sumejja Canic ◽

Núria Busquets ◽

...

Keyword(s):

Quantitative Polymerase Chain Reaction ◽

Virus Titer ◽

Mass Rearing ◽

Qpcr Assay ◽

Detection Sensitivity ◽

Infected Mosquito ◽

Female Population ◽

Total Rna ◽

Quality Control Process ◽

One Step

Abstract Background Mosquitoes are the deadliest animals in the world. Their ability to carry and spread diseases to humans causes millions of deaths every year. Due to the lack of efficient vaccines, the control of mosquito-borne diseases primarily relies on the management of the vector. Traditional control methods such as source reduction and chemical insecticides, have proven to be sufficient to prevent the proliferation and spread of mosquito populations. The sterile insect technique (SIT) is an additional control method that can be combined with other control tactics to suppress specific mosquito populations. The SIT requires the mass-rearing and release of sterile males with the aim to induce sterility in the wild female population. Samples collected from the environment for laboratory colonization, as well as the released males, should be free from mosquito-borne viruses (MBV). Therefore, efficient detection methods with defined detection limits for MBV are required. Although a one-step reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) method was developed to detect arboviruses in human and mosquito samples, its detection limit in mosquito samples has yet to be defined. Methods We evaluated the detection sensitivity of one step RT-qPCR for targeted arboviruses in large mosquito pools, using pools of non-infected mosquitoes of various sizes (165, 320 and 1600 mosquitoes) containing one infected mosquito body with defined virus titers of chikungunya virus (CHIKV), usutu virus (USUV), West Nile virus (WNV) and Zika virus (ZIKV).Results CHIK, USUV, ZIKV, and WNV virus were detected in all tested pools using the RT-qPCR assay. Moreover, in the largest mosquito pools (1600 mosquitoes), RT-qPCR was able to detect the targeted viruses using different total RNA quantities (10, 1, 0.1 ng per reaction) as a template. Correlating the virus titer with the total RNA quantity allowed the prediction of the maximum number of mosquitoes per pool in which the RT-qPCR can theoretically detect the virus infection. The corresponding equation uses a Ct value of 36 as a cut-off value for virus detection and a virus copy number of 108 for the positive mosquito body used to spike mosquito pools, to predict the possibility of detecting CHIK, USUV, ZIKV and WNV in mosquito pools composed of 5.08 x 105, 8.74 x 106, 2.33 x 107, and 5.24 x 105 mosquitoes respectively.Conclusion Mosquito borne viruses can be reliably detected by RT-qPCR assay in pools of mosquitoes exceeding 1000 specimens. This will represent an important step to expand pathogen-free colonies for mass-rearing sterile males for programmes that have an SIT component by reducing the time and the manpower needed to conduct this quality control process.

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