3D Printed Monolithic Microreactors for Real-Time Detection of Klebsiella pneumoniae and the Resistance Gene blaNDM-1 by Recombinase Polymerase Amplification

Author(s):

Ole Behrmann,Matthias Hügle,Franz Eckardt,Iris Bachmann,Cecilia Heller,Marina Schramm,Carrie Turner,Frank T Hufert,Gregory Dame

Journal:

Micromachines

Year:

2020

Abstract:

We investigate the compatibility of three 3D printing materials towards real-time recombinase polymerase amplification (rtRPA). Both the general ability of the rtRPA reaction to occur while in contact with the cured 3D printing materials as well as the residual autofluorescence and fluorescence drift in dependence on post curing of the materials is characterized. We 3D printed monolithic rtRPA microreactors and subjected the devices to different post curing protocols. Residual autofluorescence and drift, as well as rtRPA kinetics, were then measured in a custom-made mobile temperature-controlled fluorescence reader (mTFR). Furthermore, we investigated the effects of storage on the devices over a 30-day period. Finally, we present the single- and duplex rtRPA detection of both the organism-specific Klebsiella haemolysin (khe) gene and the New Delhi metallo-β-lactamase 1 (blaNDM-1) gene from Klebsiella pneumoniae. Results: No combination of 3D printing resin and post curing protocol completely inhibited the rtRPA reaction. The autofluorescence and fluorescence drift measured were found to be highly dependent on printing material and wavelength. Storage had the effect of decreasing the autofluorescence of the investigated materials. Both khe and blaNDM-1 were successfully detected by single- and duplex-rtRPA inside monolithic rtRPA microreactors printed from NextDent Ortho Clear (NXOC). The reaction kinetics were found to be close to those observed for rtRPA performed in a microcentrifuge tube without the need for mixing during amplification. Singleplex assays for both khe and blaNDM-1 achieved a limit of detection of 2.5 × 101 DNA copies while the duplex assay achieved 2.5 × 101 DNA copies for khe and 2.5 × 102 DNA copies for blaNDM-1. Impact: We expand on the state of the art by demonstrating a technology that can manufacture monolithic microfluidic devices that are readily suitable for rtRPA. The devices exhibit very low autofluorescence and fluorescence drift and are compatible with RPA chemistry without the need for any surface pre-treatment such as blocking with, e.g., BSA or PEG.

PrimerBankID	Target Pathogen	Target Gene
RPB0104	Klebsiella pneumoniae	khe
RPB0105	Klebsiella pneumoniae	blaNDM-1

Rapid detection of Mycobacterium tuberculosis based on antigen 85B via real-time recombinase polymerase amplification

Author(s):

Y Xu,P Wu,H Zhang,J Li

Journal:

Letters in applied microbiology

Year:

2021

Abstract:

Tuberculosis (TB), as a common infectious disease, still remains a severe challenge to public health. Due to the unsatisfied clinical needs of currently available diagnostic vehicles, it is desired to establish a new approach for universally detecting Mycobacterium tuberculosis. Herein, we designed a real-time recombinase polymerase amplification (RPA) technology for identifying M. tuberculosis within 20 min at 39°C via custom-designed oligonucleotide primers and probe, which could specifically target antigen 85B (Ag85B). Particularly, the primers F4-R4 produced the fastest fluorescence signal with the probe among four pairs of designed primers in the RPA assays. The optimal primers/probe combination could effectively identify M. tuberculosis with the detection limit of 4·0 copies per μl, as it could not show a positive signal for the genomic DNA from other mycobacteria or pathogens. The Ag85B-based RPA could determine the genomic DNA extracted from M. tuberculosis with high reliability (100%, 22/22). More importantly, when testing clinical sputum samples, the real-time RPA displayed an admirable sensitivity (90%, 95% CI: 80·0-96·0%) and specificity (98%, 95% CI: 89·0-100·0%) compared to traditional smear microscopy, which was similar to the assay of Xpert MTB/RIF. This real-time RPA based Ag85B provides a promising strategy for the rapid and universal diagnosis of TB.

PrimerBankID	Target Pathogen	Target Gene
RPB0116	Mycobacterium	Ag85B

Single-strand RPA for rapid and sensitive detection of SARS-CoV-2 RNA.

Author(s):

Kim, Youngeun; Yaseen, Adam B; Kishi, Jocelyn Y; Hong, Fan; Saka, Sinem K; Sheng, Kuanwei; Gopalkrishnan, Nikhil; Schaus, Thomas E; Yin, Peng;

Journal:

medRxiv.

Year:

2020

Abstract:

We report the single-strand Recombinase Polymerase Amplification (ssRPA) method, which merges the fast, isothermal amplification of RPA with subsequent rapid conversion of the double-strand DNA amplicon to single strands, and hence enables facile hybridization-based, high-specificity readout. We demonstrate the utility of ssRPA for sensitive and rapid (4 copies per 50 渭L reaction within 10 min, or 8 copies within 8 min) visual detection of SARS-CoV-2 RNA spiked samples, as well as clinical saliva and nasopharyngeal swabs in VTM or water, on lateral flow devices. The ssRPA method promises rapid, sensitive, and accessible RNA detection to facilitate mass testing in the COVID-19 pandemic.

PrimerBankID	Target Pathogen	Target Gene
RPB0076	SARS-CoV-2	\

Rapid and accurate detection of carbapenem-resistance gene by isothermal amplification in Acinetobacter baumannii

Author(s):

Shuang Liu,Guangtao Huang,Yali Gong,Xiaojun Jin,Yudan Meng,Yizhi Peng,Junning Zhao,Xiaolu Li,Qin Li

Journal:

Burns & trauma

Year:

2020

Abstract:

Background:Acinetobacter baumannii (A. baumannii) is one of the pivotal pathogens responsible for nosocomial infections, especially in patients with low immune response, and infection with carbapenem-resistant A. baumannii has been increasing in recent years. Rapid and accurate detection of carbapenem-resistance genes in A. baumannii could be of immense help to clinical staff. Methods: In this study, a 15-μL reaction system for recombinase polymerase amplification (RPA) was developed and tested. We collected 30 clinical isolates of A. baumannii from the Burn Institute of Southwest Hospital of Third Military Medical University (Army Medical University) for 6 months and tested antibiotic susceptibility using the VITEK 2 system. A. baumannii was detected based on the bla OXA-51 gene by PCR, qPCR and 15 μL-RPA, respectively. Sensitivity and specificity were evaluated. In addition, PCR and 15 μL-RPA data for detecting the carbapenem-resistance gene bla OXA-23 were comparatively assessed. Results: The detection limit of the bla OXA-51 gene by 15 μL RPA was 2.86 CFU/ml, with sensitivity comparable to PCR and qPCR. No positive amplification signals were detected in non-Acinetobacter isolates, indicating high specificity. However, only 18 minutes were needed for the 15 μL RPA assay. Furthermore, an antibiotic susceptibility test showed that up to 90% of A. baumannii strains were resistant to meropenem and imipenem; 15 μL RPA data for detecting bla OXA-23 showed that only 10% (n = 3) of A. baumannii isolates did not show positive amplification signals, and the other 90% of (n = 27) isolates were positive, corroborating PCR results. Conclusion: We demonstrated that the new 15 μL RPA assay for detecting bla OXA-23 in A. baumannii is faster and simpler than qPCR and PCR. It is a promising alternative molecular diagnostic tool for rapid and effective detection of A. baumannii and drug-resistance genes in the field and point-of-care testing.

PrimerBankID	Target Pathogen	Target Gene
RPB0082	Acinetobacter baumannii	blaOXA-51
RPB0083	Acinetobacter baumannii	bla OXA-23

An enhanced isothermal amplification assay for viral detection.

Author(s):

Qian, Jason; Boswell, Sarah A; Chidley, Christopher; Lu, Zhi-Xiang; Pettit, Mary E; Gaudio, Benjamin L; Fajnzylber, Jesse M; Ingram, Ryan T; Ward, Rebecca H; Li, Jonathan Z; Springer, Michael;

Journal:

Nat Commun

Year:

2020

Abstract:

Rapid, inexpensive, robust diagnostics are essential to control the spread of infectious diseases. Current state of the art diagnostics are highly sensitive and specific, but slow, and require expensive equipment. Here we report the development of a molecular diagnostic test for SARS-CoV-2 based on an enhanced recombinase polymerase amplification (eRPA) reaction. eRPA has a detection limit on patient samples down to 5 viral copies, requires minimal instrumentation, and is highly scalable and inexpensive. eRPA does not cross-react with other common coronaviruses, does not require RNA purification, and takes ~45鈥塵in from sample collection to results. eRPA represents a first step toward at-home SARS-CoV-2 detection and can be adapted to future viruses within days of genomic sequence availability.

PrimerBankID	Target Pathogen	Target Gene
RPB0074	SARS-CoV-2	S gene
RPB0075	SARS-CoV-2	N gene

Simultaneous Dual-Gene Diagnosis of SARS-CoV-2 Based on CRISPR/Cas9-Mediated Lateral Flow Assay

Author(s):

Xiong, Erhu; Jiang, Ling; Tian, Tian; Hu, Menglu; Yue, Huahua; Huang, Mengqi; Lin, Wei; Jiang, Yongzhong; Zhu, Debin; Zhou, Xiaoming;

Journal:

ANGEW CHEM INT EDIT

Year:

2020

Abstract:

Few methods for the detection of SARS-CoV-2 currently have the capability to simultaneously detect two genes in a single test, which is a key measure to improve detection accuracy, as adopted by the gold standard RT-qPCR method. Developed here is a CRISPR/Cas9-mediated triple-line lateral flow assay (TL-LFA) combined with multiplex reverse transcription-recombinase polymerase amplification (RT-RPA) for rapid and simultaneous dual-gene detection of SARS-CoV-2 in a single strip test. This assay is characterized by the detection of envelope (E) and open reading frame 1ab (Orf1ab) genes from cell-cultured SARS-CoV-2 and SARS-CoV-2 viral RNA standards, showing a sensitivity of 100 RNA copies per reaction (25 μL). Furthermore, dual-gene analysis of 64 nasopharyngeal swab samples showed 100 % negative predictive agreement and 97.14 % positive predictive agreement. This platform will provide a more accurate and convenient pathway for diagnosis of COVID-19 or other infectious diseases in low-resource regions

PrimerBankID	Target Pathogen	Target Gene
RPB0059	SARS-CoV-2	E gene
RPB0060	SARS-CoV-2	Orf1ab gene

Rapid detection of SARS-CoV-2 with CRISPR-Cas12a.

Author(s):

Xiong, Dan; Dai, Wenjun; Gong, Jiaojiao; Li, Guande; Liu, Nansong; Wu, Wei; Pan, Jiaqiang; Chen, Chen; Jiao, Yingzhen; Deng, Huina; Ye, Junwei; Zhang, Xuanxuan; Huang, Huiling; Li, Qianyun; Xue, Liang; Zhang, Xiuming; Tang, Guanghui; 

Journal:

PLOS BIOL.

Year:

2020

Abstract:

The recent outbreak of betacoronavirus Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which is responsible for the Coronavirus Disease 2019 (COVID-19) global pandemic, has created great challenges in viral diagnosis. The existing methods for nucleic acid detection are of high sensitivity and specificity, but the need for complex sample manipulation and expensive machinery slow down the disease detection. Thus, there is an urgent demand to develop a rapid, inexpensive, and sensitive diagnostic test to aid point-of-care viral detection for disease monitoring. In this study, we developed a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated proteins (Cas) 12a-based diagnostic method that allows the results to be visualized by the naked eye. We also introduced a rapid sample processing method, and when combined with recombinase polymerase amplification (RPA), the sample to result can be achieved in 50 minutes with high sensitivity (1-10 copies per reaction). This accurate and portable detection method holds a great potential for COVID-19 control, especially in areas where specialized equipment is not available.

PrimerBankID	Target Pathogen	Target Gene
RPB0072	SARS-CoV-2	ORF1ab
RPB0073	SARS-CoV-2	N gene

Development of a reverse transcription recombinase polymerase amplification assay for rapid and direct visual detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

Author(s):

Lau, Yee Ling; Ismail, Ilyiana Binti; Mustapa, Nur Izati Binti; Lai, Meng Yee; Tuan Soh, Tuan Suhaila; Haji Hassan, Afifah; Peariasamy, Kalaiarasu M; Lee, Yee Leng; Abdul Kahar, Maria Kahar Bador; Chong, Jennifer; Goh, Pik Pin;

Journal:

PLoS One

Year:

2021

Abstract:

Rapid diagnosis is an important intervention in managing the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) outbreak. Real time reverse transcription polymerase chain reaction (RT-qPCR) remains the primary means for diagnosing the new virus strain but it is time consuming and costly. Recombinase polymerase amplification (RPA) is an isothermal amplification assay that does not require a PCR machine. It is an affordable, rapid, and simple assay. In this study, we developed and optimized a sensitive reverse transcription (RT)-RPA assay for the rapid detection of SARS-CoV-2 using SYBR Green I and/or lateral flow (LF) strip. The analytical sensitivity and specificity of the RT-RPA assay were tested by using 10-fold serial diluted synthetic RNA and genomic RNA of similar viruses, respectively. Clinical sensitivity and specificity of the RT-RPA assay were carried out using 78 positive and 35 negative nasopharyngeal samples. The detection limit of both RPA and RT-qPCR assays was 7.659 and 5 copies/渭L RNA, respectively with no cross reactivity with other viruses. The clinical sensitivity and specificity of RT-RPA were 98% and 100%, respectively. Our study showed that RT-RPA represents a viable alternative to RT-qPCR for the detection of SARS-CoV-2, especially in areas with limited infrastructure.

PrimerBankID	Target Pathogen	Target Gene
RPB0071	SARS-CoV-2	N gene

Suitcase Lab for Rapid Detection of SARS-CoV-2 Based on Recombinase Polymerase Amplification Assay.

Author(s):

El Wahed, Ahmed Abd; Patel, Pranav; Maier, Melanie; Pietsch, Corinna; Rüster, Dana; Böhlken-Fascher, Susanne; Kissenkötter, Jonas; Behrmann, Ole; Frimpong, Michael; Diagne, Moussa Moïse; Faye, Martin; Dia, Ndongo; Shalaby, Mohamed A; Amer, Haitham; Elgamal, Mahmoud; Zaki, Ali; Ismail, Ghada; Kaiser, Marco; Corman, Victor M; Niedrig, Matthias; Landt, Olfert; Faye, Ousmane; Sall, Amadou A; Hufert, Frank T; Truyen, Uwe; Liebert, Uwe G; Weidmann, Manfred; 

Journal:

ANAL CHEM

Year:

2021

Abstract:

In March 2020, the SARS-CoV-2 virus outbreak was declared as a world pandemic by the World Health Organization (WHO). The only measures for controlling the outbreak are testing and isolation of infected cases. Molecular real-time polymerase chain reaction (PCR) assays are very sensitive but require highly equipped laboratories and well-trained personnel. In this study, a rapid point-of-need detection method was developed to detect the RNA-dependent RNA polymerase (RdRP), envelope protein (E), and nucleocapsid protein (N) genes of SARS-CoV-2 based on the reverse transcription recombinase polymerase amplification (RT-RPA) assay. RdRP, E, and N RT-RPA assays required approximately 15 min to amplify 2, 15, and 15 RNA molecules of molecular standard/reaction, respectively. RdRP and E RT-RPA assays detected SARS-CoV-1 and 2 genomic RNA, whereas the N RT-RPA assay identified only SARS-CoV-2 RNA. All established assays did not cross-react with nucleic acids of other respiratory pathogens. The RT-RPA assay's clinical sensitivity and specificity in comparison to real-time RT-PCR (n = 36) were 94 and 100% for RdRP; 65 and 77% for E; and 83 and 94% for the N RT-RPA assay. The assays were deployed to the field, where the RdRP RT-RPA assays confirmed to produce the most accurate results in three different laboratories in Africa (n = 89). The RPA assays were run in a mobile suitcase laboratory to facilitate the deployment at point of need. The assays can contribute to speed up the control measures as well as assist in the detection of COVID-19 cases in low-resource settings.

PrimerBankID	Target Pathogen	Target Gene
RPB0063	SARS-CoV-2	RdRp gene
RPB0064	SARS-CoV-2	E gene
RPB0065	SARS-CoV-2	N gene

Reverse Transcription Recombinase Polymerase Amplification Coupled with CRISPR-Cas12a for Facile and Highly Sensitive Colorimetric SARS-CoV-2 Detection.

Author(s):

Zhang, Wei S; Pan, Jianbin; Li, Feng; Zhu, Min; Xu, Mengting; Zhu, Hongyan; Yu, Yanyan; Su, Gaoxing;

Journal:

ANAL CHEM

Year:

2021

Abstract:

The outbreak of the pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) calls for an urgent unmet need for developing a facial and cost-effective detection method. The requirement of well-trained personnel and sophisticated instrument of current primary mean (reverse transcription polymerase chain reaction, RT-PCR) may hinder the practical application worldwide. In this regard, a reverse transcription recombinase polymerase amplification (RT-RPA) coupled with CRISPR-Cas12a colorimetric assay is proposed for the SARS-CoV-2 detection. The methodology we have described herein utilizes DNA-modified gold nanoparticles (AuNPs) as a universal colorimetric readout and can specifically target ORF1ab and N regions of the SARS-CoV-2 genome. After the virus genome is amplified through RT-RPA, the resulting abundant dsDNA will bind and activate Cas12a. Under trans-cleavage degradation, the capped DNA substrate will be hydrolyzed gradually from AuNPs, demonstrating a change in the surface plasmon resonance (SPR), which can be facially monitored by UV-vis absorbance spectroscopy and naked eye observation. The high amplification efficiency from RT-RPA and Cas12a trans-cleavage process bring the sensitivity of our method to 1 copy of viral genome sequence per test. Notably, under the dual variations inspecting from the isothermal amplification and Cas12a activation process, the false positive events from other beta coronavirus members can be effectively avoided and thus significantly improve the specificity. Furthermore, the reliability of this colorimetric assay is validated by standard clinical samples from the hospital laboratory department. Through integration of the inherently high sensitivity and specificity from an RPA-coupled Cas12a system with the intrinsic simplicity of AuNP-based colorimetric assay, our method increases the practical testing availability of SARS-CoV-2.

PrimerBankID	Target Pathogen	Target Gene
RPB0066	SARS-CoV-2	ORF1ab gene
RPB0067	SARS-CoV-2	N gene