Self-powered integrated microfluidic point-of-care low-cost enabling (SIMPLE) chip

Author(s):

Erh-Chia Yeh,Chi-Cheng Fu, Lucy Hu, Rohan Thakur, Jeffrey Feng, and Luke P. Lee

Journal:

Science Advances

Year:

2017

Abstract:

Portable, low-cost, and quantitative nucleic acid detection is desirable for point-of-care diagnostics; however, current polymerase chain reaction testing often requires time-consuming multiple steps and costly equipment. We report an integrated microfluidic diagnostic device capable of on-site quantitative nucleic acid detection directly from the blood without separate sample preparation steps. First, we prepatterned the amplification initiator [magnesium acetate (MgOAc)] on the chip to enable digital nucleic acid amplification. Second, a simplified sample preparation step is demonstrated, where the plasma is separated autonomously into 224 microwells (100 nl per well) without any hemolysis. Furthermore, self-powered microfluidic pumping without any external pumps, controllers, or power sources is accomplished by an integrated vacuum battery on the chip. This simple chip allows rapid quantitative digital nucleic acid detection directly from human blood samples (10 to 105 copies of methicillin-resistant Staphylococcus aureus DNA per microliter, ~30 min, via isothermal recombinase polymerase amplification). These autonomous, portable, lab-on-chip technologies provide promising foundations for future low-cost molecular diagnostic assays.

PrimerBankID	Target Pathogen	Target Gene
RPB0192	Staphylococcus aureus	\

Development of a Panel of Recombinase Polymerase Amplification Assays for Detection of Common Bacterial Urinary Tract Infection Pathogens

Author(s):

Balakrishnan Raja,Heather J. Goux, Archana Marapadaga, Sri Rajagopalan, Katerina Kourentzi, and Richard C. Willson

Journal:

Journal of Applied Microbiology

Year:

2017

Abstract:

Aims To develop and evaluate the performance of a panel of isothermal real-time recombinase polymerase amplification (RPA) assays for detection of common bacterial urinary tract infection (UTI) pathogens. Methods and Results The panel included RPAs for Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, and Enterococcus faecalis. All five RPAs required reaction times of under 12 minutes to reach their lower limit of detection of 100 genomes per reaction or less, and did not cross-react with high concentrations of non-target bacterial genomic DNA. In a 50-sample retrospective clinical study, the five-RPA assay panel was found to have a specificity of 100% (95% CI, 78%-100%) and a sensitivity of 89% (95% CI, 75%-96%) for UTI detection. Conclusions The analytical and clinical validity of RPA for the rapid and sensitive detection of common UTI pathogens was established. Significance and Impact Rapid identification of the causative pathogens of urinary tract infections (UTIs) can be valuable in preventing serious complications by helping avoid the empirical treatment necessitated by traditional urine culture’s 48–72 hour turnaround time. The routine and widespread use of RPA to supplement or replace culture-based methods could profoundly impact UTI management and the emergence of multidrug-resistant pathogens.

PrimerBankID	Target Pathogen	Target Gene
RPB0177	Klebsiella pneumoniae	khe
RPB0186	Pseudomonas aeruginosa	lasB

Rapid and visual detection of Mycobacterium tuberculosis complex using recombinase polymerase amplification combined with lateral flow strips

Author(s):

Qinglin Ma , Houming Liu , Feidi Ye , Guangxin Xiang , Wanshui Shan , Wanli Xing

Journal:

Molecular and Cellular Probes

Year:

2017

Abstract:

To definitively diagnose active pulmonary Tuberculosis (TB), Mycobacterium tuberculosis complex (MTBC) bacilli must be identified within clinical specimens from patients. In this study, we introduced a rapid and visual detection method of MTBC using recombinase polymerase amplification (RPA) combined with lateral flow (LF) strips. The LF-RPA assay, read results with naked eyes, could detect as few as 5 genome copies of M. tuberculosis H37Rv (ATCC 27294) per reaction and had no cross-reactions with other control bacteria even using excessive amount of template DNA. The system could work well at a broad range of temperature 25-45 °C and reach detectable level even within 5 min. When testing a total of 137 clinical specimens, the sensitivity and specificity of the LF-RPA assay were 100% (95% CI: 95.94%-100%) and 97.92% (95% CI: 88.93%-99.95%), respectively, compared to culture identification method. Therefore, the LF-RPA system we have demonstrated is a rapid, simple, robust method for MTBC detection which, subject to the availability of a suitable sample extraction method, has the potentiality to diagnose TB at the point-of-care testing.

PrimerBankID	Target Pathogen	Target Gene
RPB0183	Mycobacterium tuberculosis	IS1081

Quantification of viable and non-viable Legionella spp. by heterogeneous asymmetric recombinase polymerase amplification (haRPA) on a flow-based chemiluminescence microarray

Author(s):

Catharina Kober , Reinhard Niessner, Michael Seidel 2

Journal:

Biosensors & bioelectronics

Year:

2018

Abstract:

Increasing numbers of legionellosis outbreaks within the last years have shown that Legionella are a growing challenge for public health. Molecular biological detection methods capable of rapidly identifying viable Legionella are important for the control of engineered water systems. The current gold standard based on culture methods takes up to 10 days to show positive results. For this reason, a flow-based chemiluminescence (CL) DNA microarray was developed that is able to quantify viable and non-viable Legionella spp. as well as Legionella pneumophila in one hour. An isothermal heterogeneous asymmetric recombinase polymerase amplification (haRPA) was carried out on flow-based CL DNA microarrays. Detection limits of 87 genomic units (GU) µL-1 and 26GUµL-1 for Legionella spp. and Legionella pneumophila, respectively, were achieved. In this work, it was shown for the first time that the combination of a propidium monoazide (PMA) treatment with haRPA, the so-called viability haRPA, is able to identify viable Legionella on DNA microarrays. Different proportions of viable and non-viable Legionella, shown with the example of L. pneumophila, ranging in a total concentration between 101 to 105GUµL-1 were analyzed on the microarray analysis platform MCR 3. Recovery values for viable Legionella spp. were found between 81% and 133%. With the combination of these two methods, there is a chance to replace culture-based methods in the future for the monitoring of engineered water systems like condensation recooling plants.

PrimerBankID	Target Pathogen	Target Gene
RPB0178	Legionella pneumophila	16S rRNA

Handheld isothermal amplification and electrochemical detection of DNA in resource-limited settings

Author(s):

Maria-Nefeli Tsaloglou , Alex Nemiroski , Gulden Camci-Unal , Dionysios C Christodouleas , Lara P Murray , John T Connelly , George M Whitesides

Journal:

Analytical Biochemistry

Year:

2018

Abstract:

This paper demonstrates a new method for electrochemical detection of specific sequences of DNA present in trace amounts in serum or blood. This method is designed for use at the point-of-care (particularly in resource-limited settings). By combining recombinase polymerase amplification (RPA)- an isothermal alternative to the polymerase chain reaction - with an electroactive mediator, this electrochemical methodology enables accurate detection of DNA in the field using a low-cost, portable electrochemical analyzer (specifically designed for this type of analysis). This handheld device has four attributes: (1) It uses disposable, paper-based strips that incorporate screen-printed carbon electrodes; (2) It accomplishes thermoregulation with ±0.1 °C temperature accuracy; (3) It enables electrochemical detection using a variety of pulse sequences, including square-wave and cyclic voltammetry, and coulometry; (4) It is operationally simple to use. Detection of genomic DNA from Mycobacterium smegmatis (a surrogate for M. tuberculosis-the main cause of tuberculosis), and from M. tuberculosis itself down to ∼0.040 ng/μL provides a proof-of-concept for the applicability of this method of screening for disease using molecular diagnostics. With minor modifications to the reagents, this method will also enable field monitoring of food and water quality.

PrimerBankID	Target Pathogen	Target Gene
RPB0182	Mycobacterium tuberculosis	16S rRNA

Rapid visual detection of Mycobacterium avium subsp. paratuberculosis by recombinase polymerase amplification combined with a lateral flow dipstick

Author(s):

Guimin Zhao,Hongmei Wang,Peili Hou,Chengqiang He,Hongbin He

Journal:

Journal of veterinary science

Year:

2018

Abstract:

Paratuberculosis (Johne's disease) is a chronic debilitating disease of domestic and wild ruminants. However, widespread point-of-care testing is infrequent due to the lack of a robust method. The isothermal recombinase polymerase amplification (RPA) technique has applied for rapid diagnosis. Herein, RPA combined with a lateral flow dipstick (LFD) assay was developed to estimate DNA from Mycobacterium avium subsp. paratuberculosis. First, analytical specificity and sensitivity of the RPA-nfo primer and probe sets were assessed. The assay successfully detected M. paratuberculosis DNA in 30 min at 39°C with a detection limit of up to eight copies per reaction, which was equivalent to that of the real-time quantitative polymerase chain reaction (qPCR) assay. The assay was specific, as it did not amplify genomes from five other Mycobacterium spp. or five pathogenic enteric bacteria. Six hundred-twelve clinical samples (320 fecal and 292 serum) were assessed by RPA-LFD, qPCR, and enzyme-linked immunosorbent assay, respectively. The RPA-LFD assay yielded 100% sensitivity, 97.63% specificity, and 98.44% concordance rate with the qPCR results. This is the first report utilizing an RPA-LFD assay to visualize and rapidly detect M. paratuberculosis. Our results show this assay should be a useful method for the diagnosis of paratuberculosis in resource-constrained settings.

PrimerBankID	Target Pathogen	Target Gene
RPB0123	Mycobacterium avium subsp. Paratuberculosis	IS900

Automated real-time detection of drug-resistant Mycobacterium tuberculosis on a lab-on-a-disc by Recombinase Polymerase Amplification

Author(s):

I L G Law,J F C Loo,H C Kwok,H Y Yeung,C C H Leung,M Hui,S Y Wu,H S Chan,Y W Kwan,H P Ho,S K Kong

Journal:

Analytical biochemistry

Year:

2018

Abstract:

With the emergence of multi- and extensive-drug (MDR/XDR) resistant Mycobacterium tuberculosis (M. tb), tuberculosis (TB) persists as one of the world's leading causes of death. Recently, isothermal DNA amplification methods received much attention due to their ease of translation onto portable point-of-care (POC) devices for TB diagnosis. In this study, we aimed to devise a simple yet robust detection method for M. tb. Amongst the numerous up-and-coming isothermal techniques, Recombinase Polymerase Amplification (RPA) was chosen for a real-time detection of TB with or without MDR. In our platform, real-time RPA (RT-RPA) was integrated on a lab-on-a-disc (LOAD) with on-board power to maintain temperature for DNA amplification. Sputa collected from healthy volunteers were spiked with respective target M. tb samples for testing. A limit of detection of 102 colony-forming unit per millilitre in 15 min was achieved, making early detection and differentiation of M. tb strains highly feasible in extreme POC settings. Our RT-RPA LOAD platform has also been successfully applied in the differentiation of MDR-TB from H37Ra, an attenuated TB strain. In summary, a quantitative RT-RPA on LOAD assay with a high level of sensitivity was developed as a foundation for further developments in medical bedside and POC diagnostics.

PrimerBankID	Target Pathogen	Target Gene
RPB0121	Mycobacterium tuberculosis	IS6110
RPB0122	Mycobacterium tuberculosis	katG

Development and evaluation of a rapid recombinase polymerase amplification assay for the detection of human enterovirus 71

Author(s):

Dan Yin,Yanan Zhu,Kaifeng Wang,Jing Wang,Xiru Zhang,Min Han,Yaqing He,Qing Chen,Guifang Hu

Journal:

Archives of virology

Year:

2018

Abstract:

Enterovirus 71 (EV71) is one of the most common pathogens of hand, foot, and mouth disease (HFMD). A rapid reverse transcription recombinase polymerase amplification (RT-RPA) assay was established to detect EV71 subgenotype C4 (EV71-C4). The 95% detection limit of the RT-RPA was 3.767 log10 genomic copies (LGC)/reaction. The specificity was 100%. In a clinical sample evaluation, this approach demonstrated sufficient clinical performance when compared with a commercial RT-qPCR diagnostic kit. Thus, the RT-RPA assay may be a promising alternative for the detection of EV71-C4.

PrimerBankID	Target Pathogen	Target Gene
RPB0087	EV-A71	VP1 gene

Rapid and Visual Detection of Coxiella burnetii Using Recombinase Polymerase Amplification Combined with Lateral Flow Strips

Author(s):

Yong Qi,Qiong Yin,Yinxiu Shao,Suqin Li,Hongxia Chen,Wanpeng Shen,Jixian Rao,Jiameng Li,Xiaoling Li,Yu Sun,Yu Lin,Yi Deng,Wenwen Zeng,Shulong Zheng,Suyun Liu,Yuexi Li

Journal:

BioMed research international

Year:

2018

Abstract:

Coxiella burnetii, a global-distributed biological warfare agent, is the causative agent of Q fever. Correct diagnosis of Q fever is challenging and developing a fast, simple, and reliable detection method is necessary. In this study, recombinase polymerase amplification (RPA) assay combined with lateral flow (LF) test was developed targeting 23S rRNA gene of C. burnetii Xinqiao strain. Primers and probe were designed and synthesized, with one set with high amplification efficiency screened for establishment of the method. Reaction conditions were optimized. Sensitivity, specificity, and accuracy were evaluated. The established RPA-LF reaction could be completed in 30 minutes by combining RPA at 37°C with LF at room temperature, with visually judged results. The method showed good specificity without recognizing other bacteria evaluated. It detected positive plasmid and genomic DNA at levels of 10 copies/reaction and 7 copies/reaction, respectively, levels comparable to that of real-time quantitative PCR (RT-qPCR) targeting 23S rRNA gene established previously. Both RPA-LF and RT-qPCR were used to detect C. burnetii-infected mouse samples and the results were fully consistent. The method showed superior detection performance and will provide technical support against C. burnetii in resources-limited areas.

PrimerBankID	Target Pathogen	Target Gene
RPB0086	Coxiella burnetii	23S rRNA gene

Naked eye detection of the Mycobacterium tuberculosis complex by recombinase polymerase amplification-SYBR green I assays

Author(s):

Nuntita Singpanomchai,Yukihiro Akeda,Kazunori Tomono,Aki Tamaru,Pitak Santanirand,Panan Ratthawongjirakul

Journal:

Journal of clinical laboratory analysis

Year:

2019

Abstract:

Background: Rapid diagnosis of Mycobacterium tuberculosis (Mtb) is key to controlling the spread of tuberculosis, which is a global health concern. In this study, isothermal recombinase polymerase amplification (RPA) was developed to detect specific targets of Mtb, IS6110 and IS1081. Additionally, SYBR Green I was used for endpoint detection of the RPA products by the naked eye. Method: A total of 146 genomic Mtb DNA samples and 24 genomic nontuberculous mycobacteria (NTM) DNA samples were amplified at IS6110 and IS1081 by RPA. After a complete amplification, the RPA amplicons were examined by agarose gel electrophoresis (RPA-AGE) and SYBR Green I (RPA-S) assays. The performance of the RPA assays was evaluated by comparing them to a conventional PCR. Results: The RPA assay demonstrated to have a good capability to differentiate Mtb from NTM with a very short turnaround time at a constant temperature. Compared to conventional PCR, the sensitivities and specificities of RPA-AGE for IS6110 and IS1081 were 100%. The specificity of RPA-S was 100% for both targets; however, its sensitivities for IS6110 and IS1081 were 97.95% and 99.32%, respectively. The limits of detection of IS6110 RPA-AGE and RPA-S were 0.05 and 0.5 ng, respectively, while the LODs of IS1081 RPA-AGE and RPA-S were 0.00005 and 0.05 ng, respectively. Both RPA assays showed a satisfying diagnostic specificity, with no cross-reaction with other bacteria. Conclusion: A rapid, sensitive, naked eye RPA assay can be integrated into point-of-care diagnosis for Mtb detection, especially in remote areas where laboratory instrument resources are limited.

PrimerBankID	Target Pathogen	Target Gene
RPB0119	Mycobacterium tuberculosis	IS6110
RPB0120	Mycobacterium tuberculosis	IS1081