Rapid and visual detection of Staphylococcus aureus in milk using a recombinase polymerase amplification-lateral flow assay combined with immunomagnetic separation

Author(s):

Ya-Lei Wang,Xin Zhang,Quan Wang,Peng-Xuan Liu,Wei Tang,Rong Guo,Hai-Yang Zhang,Zhao-Guo Chen,Xian-Gan Han,Wei Jiang

Journal:

Journal of applied microbiology

Year:

2022

Abstract:

Aims: The aim of this study was to develop a novel approach using lateral flow recombinase polymerase amplification (RPA-LF) combined with immunomagnetic separation (IMS) for the rapid detection of Staphylococcus aureus in milk. Methods and results: Under optimum conditions, the average capture efficiency values for S. aureus strains (104 colony-forming units [CFU] per ml) was above 95.0% in PBST and ~80% in milk within 45 min with 0.7 mg immunomagnetic beads. The RPA-LF assay, which comprised DNA amplification via RPA at 39°C for 10 min and visualization of the amplicons through LF strips for 5 min, detected S. aureus within 15 min. The method only detected S. aureus and did not show cross-reaction with other bacteria, exhibiting a high level of specificity. Sensitivity experiments confirmed a detection limit of RPA-LF assay as low as 600 fg per reaction for the S. aureus genome (corresponding to approximately 36 CFU of S. aureus), which was about 16.7-fold more sensitive than that of the conventional polymerase chain reaction method. When RPA-LF was used in combination with IMS to detect S. aureus inoculated into artificially contaminated milk, it exhibited a detection limit of approximately 40 CFU per reaction. Conclusions: The newly developed IMS-RPA-LF method enabled detection of S. aureus at levels as low as 40 CFU per reaction in milk samples without culture enrichment for an overall testing time of only 70 min. Significance and impact of the study: The newly developed IMS-lateral flow RPA-LF assay effectively combines sample preparation, amplification and detection into a single platform. Because of its high sensitivity, specificity and speed, the IMS-RPA-LF assay will have important implications for the rapid detection of S. aureus in contaminated food.

PrimerBankID	Target Pathogen	Target Gene
RPB0163	Staphylococcus aureus	nuc

Development of a multi-recombinase polymerase amplification assay for rapid identification of COVID-19, influenza A and B

Author(s):

Li-Guo Liang,Miao-Jin Zhu,Rui He,Dan-Rong Shi,Rui Luo,Jia Ji,Lin-Fang Cheng,Xiang-Yun Lu,Wei Lu,Fu-Ming Liu,Zhi-Gang Wu,Nan-Ping Wu,Hang Chen,Zhe Chen,Hang-Ping Yao

Journal:

journal of medical virology

Year:

2023

Abstract:

The coronavirus disease 2019 (COVID-19) pandemic caused extensive loss of life worldwide. Further, the COVID-19 and influenza mix-infection had caused great distress to the diagnosis of the disease. To control illness progression and limit viral spread within the population, a real-time reverse-transcription PCR (RT-PCR) assay for early diagnosis of COVID-19 was developed, but detection was time-consuming (4-6 h). To improve the diagnosis of COVID-19 and influenza, we herein developed a recombinase polymerase amplification (RPA) method for simple and rapid amplification of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19 and Influenza A (H1N1, H3N2) and B (influenza B). Genes encoding the matrix protein (M) for H1N1, and the hemagglutinin (HA) for H3N2, and the polymerase A (PA) for Influenza B, and the nucleocapsid protein (N), the RNA-dependent-RNA polymerase (RdRP) in the open reading frame 1ab (ORF1ab) region, and the envelope protein (E) for SARS-CoV-2 were selected, and specific primers were designed. We validated our method using SARS-CoV-2, H1N1, H3N2 and influenza B plasmid standards and RNA samples extracted from COVID-19 and Influenza A/B (RT-PCR-verified) positive patients. The method could detect SARS-CoV-2 plasmid standard DNA quantitatively between 102 and 105 copies/ml with a log linearity of 0.99 in 22 min. And this method also be very effective in simultaneous detection of H1N1, H3N2 and influenza B. Clinical validation of 100 cases revealed a sensitivity of 100% for differentiating COVID-19 patients from healthy controls when the specificity was set at 90%. These results demonstrate that this nucleic acid testing method is advantageous compared with traditional PCR and other isothermal nucleic acid amplification methods in terms of time and portability. This method could potentially be used for detection of SARS-CoV-2, H1N1, H3N2 and influenza B, and adapted for point-of-care (POC) detection of a broad range of infectious pathogens in resource-limited settings.

PrimerBankID	Target Pathogen	Target Gene
RPB0021	SARS-CoV-2	N
RPB0022	SARS-CoV-2	E
RPB0023	SARS-CoV-2	ORF1ab
RPB0024	Influenza A virus (H1N1)	M
RPB0025	Influenza A virus (H3N2)	HA
RPB0026	Influenza B virus	PA

Computer vision enabled funnel adapted sensing tube (FAST) for power-free and pipette-free nucleic acid detection

Author(s):

Mengdi Bao,Shuhuan Zhang,Chad Ten Pas,Stephen J Dollery,Ruth V Bushnell,F N U Yuqing,Rui Liu,Guoyu Lu,Gregory J Tobin,Ke Du

Journal:

Lab On A Chip

Year:

2022

Abstract:

A simple, portable, and low-cost microfluidic system-funnel adapted sensing tube (FAST) is developed as an integrated, power-free, and pipette-free biosensor for viral nucleic acids. This FAST chip consists of four reaction chambers separated by carbon fiber rods, and the reagents in each chamber are transferred and mixed by manually removing the rods. Rather than using electrical heaters, only a hand warmer pouch is used for an isothermal recombinase polymerase amplification (RPA) and CRISPR-Cas12a reaction. The signal produced by the RPA-CRISPR reaction is observed by the naked eye using an inexpensive flashlight as a light source. The FAST chip is fabricated using water-soluble polyvinyl alcohol (PVA) as a sacrificial core, which is simple and environmentally friendly. Using a SARS-CoV-2 fragment as a target, a ∼10 fM (6 × 103 copies per μL) detection limit is achieved. To generalize standard optical readout for individuals without training, a linear kernel algorithm is created, showing an accuracy of ∼100% for identifying both positive and negative samples in FAST. This power-free, pipette-free, disposable, and simple device will be a promising tool for nucleic acid diagnostics in either clinics or low-resource settings.

PrimerBankID	Target Pathogen	Target Gene
RPB0031	SARS-CoV-2	S

Nanozyme-strip for rapid and ultrasensitive nucleic acid detection of SARS-CoV-2

Author(s):

Xiangqin Meng, Sijia Zou, Dandan Li , Jiuyang He, Ling Fang, Haojue Wang, Xiyun Yan, Demin Duan, Lizeng Gao

Journal:

Biosensors & Bioelectronics

Year:

2022

Abstract:

The coronavirus disease 2019 (COVID-19) pandemic has created a huge demand for sensitive and rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The current gold standard for SARS-CoV-2 detection is reverse transcription-polymerase chain reaction (RT-PCR)-based nucleic acid amplification. However, RT-PCR is time consuming and requires specialists and large instruments that are unattainable for point-of-care testing (POCT). To develop POCT for SARS-CoV-2, we combined recombinase polymerase amplification (RPA) and FeS2 nanozyme strips to achieve facile nucleic acid amplification and subsequent colorimetric signal enhancement based on the high peroxidase-like activity of the FeS2 nanozymes. This method showed a nucleic acid limit of detection (LOD) for SARS-CoV-2 of 200 copies/mL, close to that of RT-PCR. The unique catalytic properties of the FeS2 nanozymes enabled the nanozyme-strip to amplify colorimetric signals via the nontoxic 3,3',5,5'-tetramethylbenzidine (TMB) substrate. Importantly, the detection of clinical samples of human papilloma virus type 16 (HPV-16) showed 100% agreement with previous RT-PCR results, highlighting the versatility and reliability of this method. Our findings suggest that nanozyme-based nucleic acid detection has great potential in the development of POCT diagnosis for COVID-19 and other viral infections.

PrimerBankID	Target Pathogen	Target Gene
RPB0032	SARS-CoV-2	ORF1ab

Aptamer-based colorimetric detection of methicillin-resistant Staphylococcus aureus by using a CRISPR/Cas12a system and recombinase polymerase amplification

Author(s):

Luyu Wei,Zhilong Wang,Jia Wang,Xiaohong Wang,Yiping Chen

Journal:

Analytica chimica acta

Year:

2022

Abstract:

Detection of methicillin-resistant Staphylococcus aureus (MRSA) with superior accuracy, timeliness, and simplicity is highly valuable in clinical diagnosis and food safety. In this study, an aptamer-based colorimetric biosensor was developed to detect MRSA by using a CRISPR/Cas12a system and recombinase polymerase amplification (RPA). The aptamer of silver ion (Ag+) pre-coupled to magnetic nanoparticles was employed not only as the substrate of trans-cleavage in the CRISPR/Cas12a system, but also as the modulator of Ag+-3,3',5,5'-tetramethylbenzidine (TMB) chromogenic reaction, innovatively integrating the powerful CRISPR/Cas12a system with convenient colorimetry. The utilized aptamer containing consecutive and interrupted cytosine: cytosine mismatched base pairs also served as a signal amplifier because of the one-to-multiple binding of the aptamer to Ag+. Using triple amplification of RPA, multiple-turnover nuclease activity of Cas12a, and cytosine-Ag+-cytosine coordination chemistry, MRSA was detected as low as 8 CFU mL-1. Moreover, its satisfactory accuracy in the analysis of real samples, together with visualization and simplicity, revealed the great potential of the proposed biosensor as a robust antibiotic-resistant bacteria detection platform.

PrimerBankID	Target Pathogen	Target Gene
RPB0162	Staphylococcus aureus	mecA

Duplex real-time fluorescent recombinase polymerase amplification for the rapid and sensitive detection of two resistance genes in drug-resistant Staphylococcus aureus

Author(s):

Zhonglin Shi,Yanan Li,Anzhong Hu,Junsheng Cui,Min Shao,Ling Zhu,Ke Yang,Yong Liu,Guoqing Deng,Cancan Zhu

Journal:

Journal of microbiological methods

Year:

2022

Abstract:

In the clinic, drug-resistant Staphylococcus aureus (S. aureus) is the most common suppurative infection pathogen in humans. It can cause local infections in humans and animals, such as pneumonia, mastitis, and other systemic illnesses. At present, the detection of drug-resistant S. aureus includes traditional isolation by culture and antimicrobial susceptibility tests. However, these methods are complicated in experimental design, specialized in operation and time consuming. Therefore, a rapid and accurate drug-resistant S. aureus detection technology is urgently needed. In this study, we combined duplex pairs of fluorescent probes with recombinase polymerase amplification (RPA) to realize the simultaneous detection of two resistance genes in drug-resistant S. aureus. The method shows low detection limit, detecting 20 copies within 10 min. The analytical specificity of this method was evaluated with several related drug-resistant bacterial strains (Non-resistant S. aureus, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae), and the positive signal was only observed with drug-resistant S. aureus. In addition, the clinical suitability of this method was verified by 30 clinical isolates. Compared with qPCR, the coincidence rate of drug resistance genes were 100% (mecA) and 96.7% (ermA), respectively. These results show that the duplex real-time fluorescent RPA assay is a rapid, low detection limit and specific detection of mecA and ermA genes in isolates of drug-resistant S. aureus.

PrimerBankID	Target Pathogen	Target Gene
RPB0160	Staphylococcus aureus	mecA
RPB0161	Staphylococcus aureus	ermA

A New Auto-RPA-Fluorescence Detection Platform for SARS-CoV-2

Author(s):

Jing Tian, Biao Chen, Bin Zhang, Tantan Li, Zhiqiang Liang, Yujin Guo, Huping Jiao, Fenghong Liang, Longquan Xiang, Fanzhong Lin, Ruiwen Ren, Qingbin Liu

Journal:

Laboratory Medicine

Year:

2023

Abstract:

Objective: The outbreak of COVID-19 caused by SARS-CoV-2 has led to a serious worldwide pandemic. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)-based methods were recommended for routine detection of SARS-CoV-2 RNA. Because the reaction time and analytical sensitivity of qRT-PCR limits the diagnosis of SARS-CoV-2, development of a quick process of SARS-CoV-2 detection technology with high analytical sensitivity remains urgent. Methods: We combined isothermal amplification and fluorescence detection technology to develop a new auto-recombinase polymerase amplification (RPA)-fluorescence platform that could be used in the diagnosis of SARS-CoV-2. Results: By optimization of primers and probes, the RPA platform could detect SARS-CoV-2 nucleotides within 15 min. The limits of detection and specificity of the auto-RPA-fluorescence platform were 5 copies/µL and 100%, respectively. The accuracy of detection of the auto-RPA-fluorescence platform in the 16 positive samples was 100%. Conclusion: The RPA platform is a potential technology for the diagnosis of SARS-CoV-2 infection.

PrimerBankID	Target Pathogen	Target Gene
RPB0017	SARS-CoV-2	N
RPB0018	SARS-CoV-2	S

Rapid detection of human coronavirus NL63 by isothermal reverse transcription recombinase polymerase amplification

Author(s):

Aline Dorendorf,Iris Bachmann,Martin Spiegel,Ahmed Abd El Wahed,Gregory Dame,Frank Hufert

Journal:

Journal of clinical virology plus

Year:

2022

Abstract:

Background: Human coronaviruses are one of the leading causes for respiratory tract infections and for frequent primary care consultation. The human coronavirus NL63 (HCoV..µNL63) is one representative of the seasonal coronaviruses and capable of infecting the upper and lower respiratory tract and causative agent for croup in children. Objectives: For fast detection of HCoV-NL63, we developed an isothermal reverse transcription recombinase polymerase amplification (RT-RPA) assay. Study design: The analytical sensitivities of the RT-RPA assay were identified for in vitro transcribed ribonucleic acid (RNA) and for genomic viral RNA from cell culture supernatant. Moreover, specificity was tested with nucleic acids from other human coronaviruses and a variety of clinically relevant respiratory viruses. Finally, a clinical nasopharyngeal swab sample with spiked genomic viral HCoV-NL63 RNA was analyzed. Results: Our HCoV-NL63 RT-RPA assay is highly specific and has an analytical sensitivity of 13 RNA molecules/reaction for in vitro transcribed RNA. For genomic viral RNA from cell culture supernatant spiked into a clinical nasopharyngeal swab sample the assay...s analytical sensitivity is 170 RNA molecules/reaction. The assay shows amplification of the lowest detectable target copy number after 8 minutes and 7 minutes, respectively. Conclusions: We were able to design a sensitive and specific RT-RPA assay for the detection of HCoV-NL63. Additionally, the assay is characterized by short duration, isothermal amplification, and simple instrumentation.

PrimerBankID	Target Pathogen	Target Gene
RPB0093	HCoV-NL63	N-gene
RPB0171	HCoV-NL63	N-gene

An ultrasensitive and rapid "sample-to-answer" microsystem for on-site monitoring of SARS-CoV-2 in aerosols using "in situ" tetra-primer recombinase polymerase amplification

Author(s):

Shanglin Li, Bao Li, Xinyue Li, Ce Liu, Xiao Qi, Yin Gu, Baobao Lin, Lingli Sun, Lan Chen, Bingqian Han, Jiazhen Guo, Yanyi Huang, Shuangsheng Wu, Lili Ren, Jianbin Wang, Jingwei Bai, Jianxin Ma, Maosheng Yao, Peng Liu

Journal:

Biosensors & Bioelectronics

Year:

2022

Abstract:

Airborne transmissibility of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the urgent need for aerosol monitoring of SARS-CoV-2 to prevent sporadic outbreaks of COVID-19. The inadequate sensitivity of conventional methods and the lack of an on-site detection system limited the practical SARS-CoV-2 monitoring of aerosols in public spaces. We have developed a novel SARS-CoV-2-in-aerosol monitoring system (SIAMs) which consists of multiple portable cyclone samplers for collecting aerosols from several venues and a sensitive "sample-to-answer" microsystem employing an integrated cartridge for the analysis of SARS-CoV-2 in aerosols (iCASA) near the sampling site. By seamlessly combining viral RNA extraction based on a chitosan-modified quartz filter and "in situ" tetra-primer recombinase polymerase amplification (tpRPA) into an integrated microfluidic cartridge, iCASA can provide an ultra-high sensitivity of 20 copies/mL, which is nearly one order of magnitude greater than that of the commercial kit, and a short turnaround time of 25 min. By testing various clinical samples of nasopharyngeal swabs, saliva, and exhaled breath condensates obtained from 23 COVID-19 patients, we demonstrate that the positive rate of our system was 3.3 times higher than those of the conventional method. Combining with multiple portable cyclone samplers, we detected 52.2% (12/23) of the aerosol samples, six times higher than that of the commercial kit, collected from the isolation wards of COVID-19 patients, demonstrating the excellent performance of our system for SARS-CoV-2-in-aerosol monitoring. We envision the broad application of our microsystem in aerosol monitoring for fighting the COVID-19 pandemic.

PrimerBankID	Target Pathogen	Target Gene
RPB0036	SARS-CoV-2	ORF1ab

Recombinase Polymerase Amplification Combined with Fluorescence Immunochromatography Assay for On-Site and Ultrasensitive Detection of SARS-CoV-2

Author(s):

Guangyu Wang , Xingsheng Yang, Hao Dong, Zhijie Tu , Yong Zhou, Zhen Rong , Shengqi Wang

Journal:

Pathogens

Year:

2022

Abstract:

This study established a portable and ultrasensitive detection method based on recombinase polymerase amplification (RPA) combined with high-sensitivity multilayer quantum dot (MQD)-based immunochromatographic assay (ICA) to detect the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The RPA-MQD-based ICA method is reported for the first time and has the following advantages: (i) RPA is free from the constraints of instruments and can be promoted in point-of-care testing (POCT) scenarios, (ii) fluorescence ICA enhances the portability of detection operation so that the entire operation time is controlled within 1 h, and (iii) compared with common colorimetric-based RPA-ICA, the proposed assay used MQD to provide strong and quantifiable fluorescence signal, thus enhancing the detection sensitivity. With this strategy, the proposed RPA-MQD-based ICA can amplify and detect the SARS-CoV-2 nucleic acid on-site with a sensitivity of 2 copies/reaction, which is comparable to the sensitivity of commercial reverse transcription quantitative polymerase chain reaction (RT-qPCR) kits. Moreover, the designed primers did not cross-react with other common respiratory viruses, including adenovirus, influenza virus A, and influenza virus B, suggesting high specificity. Thus, the established portable method can sensitively detect SARS-CoV-2 nucleic acid without relying on equipment, having good application prospects in SARS-CoV-2 detection scenarios under non-lab conditions.

PrimerBankID	Target Pathogen	Target Gene
RPB0035	SARS-CoV-2	S