A multi-country phase 2 study to evaluate the suitcase lab for rapid detection of SARS-CoV-2 in seven Sub-Saharan African countries: Lessons from the field

Author(s):

Arianna Ceruti,Ndongo Dia,Adeleye Solomon Bakarey,Judah Ssekitoleko,Soa Fy Andriamandimby,Padra Malwengo-Kasongo,Rasheeda H A Ahmed,Rea Maja Kobialka,Jean Michel Heraud,Moussa Moise Diagne,Marie Henriette Dior Dione,Idrissa Dieng,Martin Faye,Ousmane Faye,Jean Théophile Rafisandratantsoa,Jean-Pierre Ravalohery,Claudio Raharinandrasana,Tsiry H Randriambolamanantsoa,Norosoa Razanajatovo,Iony Razanatovo,Joelinotahina H Rabarison,Phillipe Dussart,Louis Kyei-Tuffuor,Abigail Agbanyo,Olubusuyi Moses Adewumi,Adeola Fowotade,Muideen Kolawole Raifu,Patient Okitale-Talunda,Gracia Kashitu-Mujinga,Christelle Mbelu-Kabongo,Steve Ahuka-Mundeke,Anguy Makaka-Mutondo,Enas M Abdalla,Sanaa M Idris,Wisal A Elmagzoub,Rahma H Ali,Eman O M Nour,Rasha S M Ebraheem,Huda H H Ahmed,Hamadelniel E Abdalla,Musab Elnegoumi,Izdihar Mukhtar,Muatsim A M Adam,Nuha Y I Mohamed,Shahinaz A Bedri,Hamdan Mustafa Hamdan,Magid Kisekka,Monica Mpumwiire,Sharley Melissa Aloyo,Joanita Nabwire Wandera,Andrew Agaba,Rogers Kamulegeya,Hosea Kiprotich,David Patrick Kateete,Paul Kadetz,Uwe Truyen,Kamal H Eltom,Anavaj Sakuntabhai,Julius Boniface Okuni,Sheila Makiala-Mandanda,Vincent Lacoste,George Olusegun Ademowo,Michael Frimpong,Amadou Alpha Sall,Manfred Weidmann,Ahmed Abd El Wahed

Journal:

Journal of Clinical Virology

Year:

2023

Abstract:

Background: The COVID-19 pandemic led to severe health systems collapse, as well as logistics and supply delivery shortages across sectors. Delivery of PCR related healthcare supplies continue to be hindered. There is the need for a rapid and accessible SARS-CoV-2 molecular detection method in low resource settings. Objectives: To validate a novel isothermal amplification method for rapid detection of SARS-CoV-2 across seven sub-Sharan African countries. Study design: In this multi-country phase 2 diagnostic study, 3,231 clinical samples in seven African sites were tested with two reverse transcription Recombinase-Aided Amplification (RT-RAA) assays (based on SARS-CoV-2 Nucleocapsid (N) gene and RNA-dependent RNA polymerase (RdRP) gene). The test was performed in a mobile suitcase laboratory within 15 min. All results were compared to a real-time RT-PCR assay. Extraction kits based on silica gel or magnetic beads were applied. Results: Four sites demonstrated good to excellent agreement, while three sites showed fair to moderate results. The RdRP gene assay exhibited an overall PPV of 0.92 and a NPV of 0.88. The N gene assay exhibited an overall PPV of 0.93 and a NPV 0.88. The sensitivity of both RT-RAA assays varied depending on the sample Ct values. When comparing sensitivity between sites, values differed considerably. For high viral load samples, the RT-RAA assay sensitivity ranges were between 60.5 and 100% (RdRP assay) and 25 and 98.6 (N assay). Conclusion: Overall, the RdRP based RT-RAA test showed the best assay accuracy. This study highlights the challenges of implementing rapid molecular assays in field conditions. Factors that are important for successful deployment across countries include the implementation of standardized operation procedures, in-person continuous training for staff, and enhanced quality control measures.

PrimerBankID	Target Pathogen	Target Gene
RPB0437	SARS-CoV-2	N
RPB0438	SARS-CoV-2	RdRP

A Novel Sample-to-Answer Visual Nucleic Acid Detection System for Adenovirus Detection

Author(s):

Kui Sun,Xiaodong Yang,Yanan Wang,Qun Guan,Wenliang Fu,Chao Zhang,Qin Liu,Wenzheng An,Yongqi Zhao,Weiwei Xing,Donggang Xu

Journal:

Microbiology Spectrum

Year:

2023

Abstract:

Human adenoviruses (HAdVs) are common viruses that can cause local outbreaks in schools, communities and military camps, posing a huge threat to public health. An ideal POCT device for adenovirus detection in resource-limited settings is critical to control the spread of the virus. In this study, we developed an integrated and electricity-independent sample-to-answer system that can complete nucleic acid extraction, amplification, and detection at room temperature. This system is suitable for field and on-site detection because of its rapidity, sensitivity, lack of contamination, and lack of requirements of high-precision instruments and skilled technicians. It consists of two separate modules, ALP FINA (alkaline lysis with the paper-based filtration isolation of nucleic acid) and SV RPA (sealed and visual recombinase polymerase amplification). The extraction efficiency of ALP FINA can reach 48 to 84%, which is close to that of the conventional centrifuge column. The detection sensitivity of SV RPA is close to 10 copies/μL of AdvB and AdvE without aerosol contamination after repeated operations. When SV RPA was applied to the detection of nasopharyngeal swab samples of 19 patients who were infected with AdvB or AdvE as well as 10 healthy volunteers, its sensitivity and specificity reached 100%, respectively. IMPORTANCE HAdV infections are readily transmittable and, in some instances, highly contagious. Early and rapid diagnosis is essential for disease control. In this work, we developed a portable, disposable, and modularized sample-to-answer detection system for AdvB and AdvE, which rendered the entire test to be completely independent of electricity and other laboratory infrastructure. Thus, this detection system can be applied in resource-limited settings, and it has the potential to be further developed as an early diagnosis method in the field.

PrimerBankID	Target Pathogen	Target Gene
RPB0419	HAdV	AdvB AdvE

CDetection.v2: One-pot assay for the detection of SARS-CoV-2

Author(s):

Xinge Wang,Yangcan Chen,Xuejia Cheng,Si-Qi Wang,Yanping Hu,Yingmei Feng,Ronghua Jin,Kangping Zhou,Ti Liu,Jianxing Wang,Kai Pan,Bing Liu,Jie Xiang,Yanping Wang,Qi Zhou,Ying Zhang,Weiye Pan,Wei Li

Journal:

Frontiers in Microbiology

Year:

2023

Abstract:

Introduction: The ongoing 2019 coronavirus disease pandemic (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and its variants, is a global public health threat. Early diagnosis and identification of SARS-CoV-2 and its variants plays a critical role in COVID-19 prevention and control. Currently, the most widely used technique to detect SARS-CoV-2 is quantitative reverse transcription real-time quantitative PCR (RT-qPCR), which takes nearly 1 hour and should be performed by experienced personnel to ensure the accuracy of results. Therefore, the development of a nucleic acid detection kit with higher sensitivity, faster detection and greater accuracy is important. Methods: Here, we optimized the system components and reaction conditions of our previous detection approach by using RT-RAA and Cas12b. Results: We developed a Cas12b-assisted one-pot detection platform (CDetection.v2) that allows rapid detection of SARS-CoV-2 in 30 minutes. This platform was able to detect up to 5,000 copies/ml of SARS-CoV-2 without cross-reactivity with other viruses. Moreover, the sensitivity of this CRISPR system was comparable to that of RT-qPCR when tested on 120 clinical samples. Discussion: The CDetection.v2 provides a novel one-pot detection approach based on the integration of RT-RAA and CRISPR/Cas12b for detecting SARS-CoV-2 and screening of large-scale clinical samples, offering a more efficient strategy for detecting various types of viruses.

PrimerBankID	Target Pathogen	Target Gene
RPB0405	SARS-CoV-2	RdRp

CRISPR\Cas9-based point-of-care lateral flow biosensor with improved performance for rapid and robust detection of Mycoplasma pneumonia

Author(s):

Rong Zhu,Han Jiang,Chaoyou Li,Yan Li,Min Peng,Junfeng Wang,Qian Wu,Chao Yan,Qingli Bo,Jie Wang,Chenlin Shen,Panzhu Qin

Journal:

Analytica Chimica Acta

Year:

2023

Abstract:

Screening of acute respiratory infections causes serious challenges in urgent point-of-care scenarios where conventional methods are impractical and alternative techniques suffer from low accuracy, poor robustness, and reliance on sophisticated instruments. As an improvement to this paradigm, we report a point-of-care lateral flow biosensor (LFB) based on the recognition property of clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (Cas9) and apply it to the detection of Mycoplasma pneumoniae (M. pneumoniae). The designed biosensor employs CRISPR/Cas9 for secondary recognition after preamplification of target gene using specific primer set, avoiding false positives caused by nontarget factors. The high amplification efficiency and low applicable temperatures of recombinase polymerase amplification brings the detection limit of the biosensor to 3 copies even at a preamplification temperature of 25 °C. Its practical application is further demonstrated with 100% accuracy by testing with 43 M. pneumoniae-infected specimens and 80 uninfected specimens. Additionally, the entire detection, including pretreatment, preamplification, CRISPR/Cas9 recognition, and visual analysis, can be completed in 30 min. Featured with the combination of CRISPR/Cas9 and LFB, the biosensor we developed herein ensures excellent convenience, accuracy, and robustness, which endows promising point-of-care screening potential for infectious pathogens.

PrimerBankID	Target Pathogen	Target Gene
RPB0432	Mycoplasma pneumoniae	\

Droplet digital recombinase polymerase amplification for multiplexed detection of human coronavirus

Author(s):

Ji Wook Choi, Won Ho Seo, Taejoon Kang, Taewook Kang, Bong Geun Chung

Journal:

Lab On A Chip

Year:

2023

Abstract:

Since the outbreak of coronavirus 2019 (COVID-19), detection technologies have been attracting a great deal of attention in molecular diagnosis applications. In particular, the droplet digital PCR (ddPCR) has become a promising tool as it offers absolute quantification of target nucleic acids with high specificity and sensitivity. In recent years, the combination of the isothermal amplification strategies has made ddPCR a popular method for on-site testing by enabling amplification at a constant temperature. However, the current isothermal ddPCR assays are still challenging due to inherent non-specific amplification. In this paper, we present a multiplexed droplet digital recombinase polymerase amplification (MddRPA) with precise initiation of the reaction. First, the reaction temperature and dynamic range of reverse transcription (RT) and RPA were characterized by real-time monitoring of fluorescence intensities. Using a droplet-based microfluidic chip, the master mix and the initiator were fractionated and rapidly mixed within well-confined droplets. Due to the high heat transfer and mass transfer of the droplets, the precise initiation of the amplification was enabled and the entire assay could be conducted within 30 min. The concentrations of target RNA in the range from 5 copies per μL to 2500 copies per μL could be detected with high linearity (R2 > 0.999). Furthermore, the multiplexed detection of three types of human coronaviruses was successfully demonstrated with high specificity (>96%). Finally, we compared the performance of the assay with a commercial RT-qPCR system using COVID-19 clinical samples. The MddRPA assay showed a 100% concordance with the RT-qPCR results, indicating its reliability and accuracy in detecting SARS-CoV-2 nucleic acids in clinical samples. Therefore, our MddRPA assay with rapid detection, precise quantification, and multiplexing capability would be an interesting method for molecular diagnosis of viral infections.

PrimerBankID	Target Pathogen	Target Gene
RPB0006	HCoV-229E	\
RPB0007	HCoV-OC43	\
RPB0008	HCoV-NL63	\

Glow-in-the-Dark Infectious Disease Diagnostics Using CRISPR-Cas9-Based Split Luciferase Complementation

Author(s):

Harmen J van der Veer, Eva A van Aalen , Claire M S Michielsen, Eva T L Hanckmann, Jeroen Deckers, Marcel M G J van Borren, Jacky Flipse, Anne J M Loonen, Joost P H Schoeber, Maarten Merkx

Journal:

ACS Central Science

Year:

2023

Abstract:

Nucleic acid detection methods based on CRISPR and isothermal amplification techniques show great potential for point-of-care diagnostic applications. However, most current methods rely on fluorescent or lateral flow assay readout, requiring external excitation or postamplification reaction transfer. Here, we developed a bioluminescent nucleic acid sensor (LUNAS) platform in which target dsDNA is sequence-specifically detected by a pair of dCas9-based probes mediating split NanoLuc luciferase complementation. LUNAS is easily integrated with recombinase polymerase amplification (RPA), providing attomolar sensitivity in a rapid one-pot assay. A calibrator luciferase is included for a robust ratiometric readout, enabling real-time monitoring of the RPA reaction using a simple digital camera. We designed an RT-RPA-LUNAS assay that allows SARS-CoV-2 RNA detection without the need for cumbersome RNA isolation and demonstrated its diagnostic performance for COVID-19 patient nasopharyngeal swab samples. Detection of SARS-CoV-2 from samples with viral RNA loads of ∼200 cp/μL was achieved within ∼20 min, showing that RPA-LUNAS is attractive for point-of-care infectious disease testing. Nucleic acid detection methods based on CRISPR and isothermal amplification techniques show great potential for point-of-care diagnostic applications. However, most current methods rely on fluorescent or lateral flow assay readout, requiring external excitation or postamplification reaction transfer. Here, we developed a bioluminescent nucleic acid sensor (LUNAS) platform in which target dsDNA is sequence-specifically detected by a pair of dCas9-based probes mediating split NanoLuc luciferase complementation. LUNAS is easily integrated with recombinase polymerase amplification (RPA), providing attomolar sensitivity in a rapid one-pot assay. A calibrator luciferase is included for a robust ratiometric readout, enabling real-time monitoring of the RPA reaction using a simple digital camera. We designed an RT-RPA-LUNAS assay that allows SARS-CoV-2 RNA detection without the need for cumbersome RNA isolation and demonstrated its diagnostic performance for COVID-19 patient nasopharyngeal swab samples. Detection of SARS-CoV-2 from samples with viral RNA loads of ∼200 cp/μL was achieved within ∼20 min, showing that RPA-LUNAS is attractive for point-of-care infectious disease testing.

PrimerBankID	Target Pathogen	Target Gene
RPB0009	SARS-CoV-2	ORF1a_L
RPB0010	SARS-CoV-2	ORF1a_S
RPB0011	SARS-CoV-2	N1_L
RPB0012	SARS-CoV-2	N1_S

A rapid and highly sensitive multiple detection of human adenovirus type 3, type 7 and respiratory syncytial virus by recombinase-aided reverse transcription PCR

Author(s):

Guohao Fan,Xiaozhou He,Ruiqing Zhang,Fengyu Tian,Xiuli Sun,Mengyi Zhang,Fengyu Li,Xinxin Shen,Xuejun Ma

Journal:

Journal of Clinical Laboratory Analysis

Year:

2023

Abstract:

Background: Polymerase chain reaction (PCR) has been widely used for many pathogen detection. However, PCR technology still suffers from long detection time and insufficient sensitivity. Recombinase-aided amplification (RAA) is a powerful nucleic acid detection tool with high sensitivity and amplification efficiency, but its complex probes and inability of multiplex detection hinder the further application of this technology. Methods: In this study, we developed and validated the multiplex reverse transcription recombinase-aided PCR (multiplex RT-RAP) assay for human adenovirus 3 (HADV3), human adenovirus 7 (HADV7), and human respiratory syncytial virus (HRSV) within 1 h with Human RNaseP protein as a reference gene to monitor the whole process. Results: Using recombinant plasmids, the sensitivity of multiplex RT-RAP for the detection of HADV3, HADV7, and HRSV was 18, 3, and 18 copies per reaction, respectively. The multiplex RT-RAP showed no cross-reactivity with other respiratory viruses, demonstrating its good specificity. A total of 252 clinical specimens were tested by multiplex RT-RAP and the results were found to be consistent with those of corresponding RT-qPCR assays. After testing serial dilutions of selected positive specimens, the detection sensitivity of multiplex RT-RAP was two to eightfold higher than that of corresponding RT-qPCR. Conclusion: We conclude the multiplex RT-RAP is a robust, rapid, highly sensitive, and specific assay with the potential to be used in the screening of clinical samples with low viral load.

PrimerBankID	Target Pathogen	Target Gene
RPB0422	HAdV3	\
RPB0423	HAdV7	\
RPB0424	HRSV A	\

RNA extraction-free workflow integrated with a single-tube CRISPR-Cas-based colorimetric assay for rapid SARS-CoV-2 detection in different environmental matrices

Author(s):

Yuliang Kang, Jiali Wang, Wensi Zhang, Yuhang Xu, Bohui Xu, Guangbo Qu, Yanyan Yu, Bing Yan, Gaoxing Su

Journal:

Journal Of Hazardous Materials

Year:

2023

Abstract:

On-site environmental surveillance of viruses is increasingly important for infection prevention and pandemic control. Herein, we report a facile single-tube colorimetric assay for detecting severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) from environmental compartments. Using glycerol as the phase separation additive, reverse transcription recombinase polymerase amplification (RT-RPA), CRISPR-Cas system activation, G-quadruplex (G4) cleavage, and G4-based colorimetric reaction were performed in a single tube. To further simplify the test, viral RNA genomes used for the one-tube assay were obtained via acid/base treatment without further purification. The whole assay from sampling to visual readout was completed within 30 min at a constant temperature without the need for sophisticated instruments. Coupling the RT-RPA to CRISPR-Cas improved the reliability by avoiding false positive results. Non-labeled cost-effective G4-based colorimetric systems are highly sensitive to CRISPR-Cas cleavage events, and the proposed assay reached the limit of detection of 0.84 copies/µL. Moreover, environmental samples from contaminated surfaces and wastewater were analyzed using this facile colorimetric assay. Given its simplicity, sensitivity, specificity, and cost-effectiveness, our proposed colorimetric assay is highly promising for applications in on-site environmental surveillance of viruses.

PrimerBankID	Target Pathogen	Target Gene
RPB0003	SARS-CoV-2	N

CRISPR Cas12a-enabled biosensors coupled with commercial pregnancy test strips for the visible point-of-care testing of SARS-CoV-2 Mycoplasma pneumonia

Author(s):

Peijie Shen,Zhenjun Si,Di Huang,Zhipeng Xu,Ziyi Wang,Mengjun Fang,Zhinan Xu

Journal:

Analyst

Year:

2023

Abstract:

The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has promoted the development of nucleic acid diagnosis technology. Several platforms with isothermal amplification methods have achieved sensitive and specific detection of SARS-CoV-2. However, they still suffer from complicated operations, delicate instruments, and unintuitive signal output modes. Here, a system consisting of CRISPR Cas12a-based biosensors and commercial pregnancy test strips (CRISPR-PTS) was established for the point-of-care testing of SARS-CoV-2. The target viral nucleic acids were finally reflected on the test strips through four steps, namely sample pretreatment, RT-RAA amplification, CRISPR Cas12a reaction, and separation-free hCG detection. This CRISPR-PTS assay possessed an outstanding sensitivity of as low as 1 copy per μL for SARS-CoV-2 detection and showed an excellent specificity in distinguishing the SARS-CoV-2 pseudovirus as well as other SARS-like viral clinical samples. In addition, the CRISPR-PTS assay performed well in practical applications, with 96.3% agreement versus RT-qPCR in spiked samples. With the advantages of low reagent cost, simple operation procedure, and visible signal output, CRISPR-PTS assay was expected to provide a strong supplement in the prevention and early diagnosis of infectious diseases in resource-limited situations.

PrimerBankID	Target Pathogen	Target Gene
RPB0433	SARS-CoV-2	E gene
RPB0434	MERS-CoV	ORF1a
RPB0435	MERS-CoV	upE
RPB0436	MERS-CoV	ORF1b

Rapid detection of Nipah virus using the one-pot RPA-CRISPR\Cas13a assay

Author(s):

Jing Miao,Lulu Zuo,Dongmei He,Zhixin Fang,Nicolas Berthet,Chao Yu,Gary Wong

Journal:

Virus Research

Year:

2023

Abstract:

Nipah virus (NiV) is a zoonotic pathogen with airborne transmission and high case fatality rates in humans. There is currently no treatment or vaccine against NiV infection approved for humans or animals, therefore early diagnosis is the key to control any potential outbreaks. In this study, we developed an optimized one-pot assay using recombinase polymerase amplification (RPA) coupled to CRISPR/Cas13a for the molecular detection of NiV. The one-pot RPA-CRISPR/Cas13a assay for NiV detection was specific and did not cross-react against other selected (re)-emerging pathogens. The sensitivity of the one-pot RPA-CRISPR/Cas13a assay for NiV detection can detect as little as 103 cp/μL of total synthetic NiV cDNA. The assay was then validated with simulated clinical samples. The results for the one-pot RPA-CRISPR/Cas13a assay could be visualized with either fluorescence or lateral flow strips for convenient clinical or field diagnostics, providing a useful supplement to the gold-standard qRT-PCR assay for detecting NiV detections.

PrimerBankID	Target Pathogen	Target Gene
RPB0384	Nipah virus	N gene