Advanced One-Pot RPA-CRISPR\Cas12a Reaction with Glycerol and Betaine for High-Sensitivity Diagnosis of mecA-Carrying Strains in Clinical Samples

Author(s):

Jingyuan Wang,Dan Wang,Linlin Fan,Xin Ye,Jian Hu,Xiaoqin Wang

Journal:

ACS Omega

Year:

2025

Abstract:

The mecA gene confers methicillin resistance in both MRSA and MR-CoNS by encoding the PBP2a protein and poses a significant public health threat due to its resistance to beta-lactam antibiotics. Rapid and accurate detection of mecA is critical for timely treatment, reducing morbidity, and preventing its spread in healthcare settings. In this study, we developed an advanced one-pot recombinase polymerase amplification (RPA)-CRISPR/Cas12a system, enhanced with glycerol and betaine, for ultrasensitive detection of the mecA gene. Glycerol's viscosity effect prevents premature interaction between Cas12a and early amplification products, while betaine enhances nucleic acid amplification. The assay demonstrated superior sensitivity, detecting as low as 5 copies/μL of mecA DNA within 60 min. Specificity testing against a panel of bacterial species confirmed the high selectivity of the assay for mecA-carrying strains with negligible cross-reactivity. Furthermore, this method exhibited excellent performance across various clinical samples, including blood, urine, and bronchoalveolar lavage fluid. Our findings underscore the potential of this advanced RPA-CRISPR/Cas12a assay as a powerful diagnostic tool for rapid, cost-effective, and highly sensitive mecA detection, offering a promising solution for clinical diagnostics and infection control.

PrimerBankID	Target Pathogen	Target Gene
RPB0308	Staphylococcus aureus	mecA gene

Establishment and Application of Duplex Recombinase-Aided Amplification Combined with Lateral Flow Dipsticks for Rapid and Simultaneous Visual Detection of Klebsiella pneumoniae and Staphylococcus aureus in Milk

Author(s):

Ni Zhang,Laiwang Hou,Darong Li,Weiqing Lan,Yong Zhao,Xiaohong Sun

Journal:

Foods

Year:

2025

Abstract:

Staphylococcus aureus and Klebsiella pneumoniae are significant and prevalent pathogens associated with bovine mastitis on dairy farms worldwide, resulting in severe infections in both dairy cows and, subsequently, human beings. Fast and dependable pathogen diagnostics are essential to minimize the effects of cow mastitis and human infections. The aim of this research was to develop a duplex recombinase-aided amplification (RAA) combined with the lateral flow dipstick (LFD) method, which was used for rapid, simultaneous detection of S. aureus and K. pneumoniae. The SKII culture medium for S. aureus and K. pneumoniae cocultivation was developed in this study. By optimizing the duplex RAA-LFD reaction conditions in terms of primer concentration, amplification temperature, and reaction time, the duplex RAA-LFD assay could successfully detect S. aureus and K. pneumoniae when the reaction was conducted at 39 °C for 20 min. The duplex RAA-LFD method demonstrated good specificity, exhibiting no cross-reactivity with other pathogens. In addition, the detection limit of the duplex RAA-LFD for S. aureus and K. pneumoniae was 60 fg of genomic DNA and 1.78 × 103 and 2.46 × 103 CFU/mL of bacteria in pure culture. Moreover, the duplex RAA-LFD technique is capable of identifying S. aureus and K. pneumoniae in artificially spiked milk samples even at very low initial concentrations of 1.78 × 101 and 2.46 × 100 CFU/mL, respectively, after 6 h of enrichment. The result of the actual samples showed that the total concordance rate of the duplex RAA-LFD method with the biochemical identification method and PCR method could reach 92.98~98.25% with high consistency. The results of this study indicated that the duplex RAA-LFD assay, which is a precise, sensitive, and simple field testing technique, can be used to identify S. aureus and K. pneumoniae and is expected to be used for disease diagnosis.

PrimerBankID	Target Pathogen	Target Gene
RPB0285	Staphylococcus aureus	nuc gene
RPB0286	Klebsiella pneumoniae	rcsA gene

Space-coded microchip for multiplexed respiratory virus detection via CRISPR-Cas12a and RPA

Author(s):

Mengqiu Xiong,Yao Wang,Shuhan Lu,Nasifu Lubanga,Tao Li,Zhihao Li,Bangshun He,Ying Li

Journal:

Talanta

Year:

2025

Abstract:

Multiple infections of respiratory viruses are common in patients with clinical respiratory diseases, but current detection methods still have problems such as complex equipment and long detection time. Rapid, low-cost, and on-site detection of human respiratory viruses is crucial for both clinical diagnosis and population screening. In this research, we created a space-coded microfluidic chip (SC-Chip) for the recognition of nine respiratory viruses: influenza A virus, influenza B virus, severe acute respiratory syndrome coronavirus 2, human coronavirus OC43, human coronavirus NL63, human coronavirus HKU1, human respiratory syncytial virus, human parainfluenza virus, and human metapneumovirus. For the first time, a comprehensive sequence comparison among these viruses was performed to design the recombinase polymerase amplification (RPA) primers and Cas12a-crRNAs. The SC-Chip partitions samples amplified by RPA into spatially coded wells preloaded with CRISPR-Cas12a detection reagents, enabling the identification of all nine viral targets in a single test using a single fluorescence probe. The chip-based assay displays 9 respiratory viruses in less than 40 min with a minimum detection limit at a concentration of 10-18 M (∼1 copy/reaction). Additionally, the efficacy of the method was assessed through its application to 35 clinical patient samples identified as being at risk for respiratory virus infection, yielding a sensitivity of 90 % and a specificity of 100 %. In summary, this space-coded microfluidic CRISPR system offers several advantages, including ease of operation, cost-effectiveness, and rapid data acquisition, thereby holding great potential for multiplexed detection of nucleic acid targets in a clinical setting.

PrimerBankID	Target Pathogen	Target Gene
RPB0254	Influenza A virus	PB1
RPB0255	Influenza B virus	PB1
RPB0256	SARS-CoV-2	ORF1ab
RPB0257	HCoV-OC43	ORF1ab
RPB0258	HCoV-NL63	ORF1ab
RPB0259	HCoV-HKU1	ORF1ab
RPB0260	HRSV A	M
RPB0261	HPIV-3	M
RPB0262	HMPV	F

A Streamlined Point-of-Care CRISPR Test for Tuberculosis Detection Directly from Sputum

Author(s):

Owen R S Dunkley,Alexandra G Bell,Nisha H Modi,Yujia Huang,Soleil Tseng,Robert Reiss,Naranjargal Daivaa,J Lucian Davis,Deninson Alejandro Vargas,Padmapriya Banada,Yingda L Xie,Cameron Myhrvold

Journal:

medRxiv.

Year:

2025

Abstract:

Mycobacterium tuberculosis (Mtb) is a major threat to global health and is responsible for over one million deaths each year. To stem the tide of cases and maximize opportunities for early interventions, there is an urgent need for affordable and simple means of tuberculosis diagnosis in under-resourced areas. We sought to develop a CRISPR-based isothermal assay coupled with a compatible, straightforward sample processing technique for point-of-care use. Here, we combine Recombinase Polymerase Amplification (RPA) with Cas13a and Cas12a, to create two parallelised one-pot assays that detect two conserved elements of Mtb (IS6110 and IS1081) and an internal control targeting human DNA. These assays were shown to be compatible with lateral flow and can be readily lyophilized. Our finalized assay exhibited sensitivity over a wide range of bacterial loads (105 to 102 CFU/mL) in sputum. The limit of detection (LoD) of the assay was determined to be 69.0 (51.0 - 86.9) CFU/mL for Mtb strain H37Rv spiked in sputum and 80.5 (59.4 - 101.6) CFU/mL for M. bovis BCG. Our assay showed no cross reactivity against a wide range of bacterial/fungal isolates. Clinical tests on 13 blinded sputum samples revealed 100% (6/6) sensitivity and 100% (7/7) specificity compared to culture. Our assay exhibited comparable sensitivity in clinical samples to the microbiological gold standard, TB culture, and to the nucleic acid state-of-the-art, GeneXpert MTB/RIF Ultra. This technology streamlines TB diagnosis from sample extraction to assay readout in a rapid and robust format, making it the first test to combine amplification and detection while being compatible with both lateral flow and lyophilization.

PrimerBankID	Target Pathogen	Target Gene
RPB0281	Mycobacterium tuberculosis	IS6110
RPB0282	Mycobacterium tuberculosis	IS1081

Establishment of an RPA-CRISPR\Cas12a combined diagnostic system for Pneumocystis jirovecii pneumonia

Author(s):

Yun Wu,Yuhan Shao,Wei Li,Ying Yu,Xia Rao,Jingyi Li,Nicholas R Waterfield,Guowei Yang

Journal:

PLOS Neglected Tropical Diseases

Year:

2025

Abstract:

Pneumocystis jirovecii causes severe pneumonia in immunocompromised individuals, leading to high mortality and an economic burden. There is a need for early detection methods suitable for low-resource settings and rapid point-of-care diagnostics. This study developed a detection method using Recombinase Polymerase Amplification (RPA) followed by CRISPR/Cas12a with fluorescence detection. The RPA primers and CRISPR-derived RNAs (crRNAs) were specifically designed to target the mitochondrial small subunit rRNA (mtSSU rRNA) gene of P. jirovecii. A total of 83 clinical samples were tested using this method, including 39 confirmed and 44 suspected cases of P. jirovecii infection. The combination of crRNA5 and crRNA6 demonstrated higher sensitivity compared to the current real-time PCR detection method, with a limit of detection (LOD) of 1 copy per reaction and showed no cross-reactions with other respiratory pathogens. The concordance of this method was validated with both infected and non-infected patients. In conclusion, the method developed in this study potentially provides a highly sensitive and rapid tool suitable for the early and on-site detection of P. jirovecii pneumonia. Furthermore, this method holds potential applications for the detection of other human pathogens, representing a significant advancement in diagnostic capabilities for low-resource settings.

PrimerBankID	Target Pathogen	Target Gene
RPB0280	Pneumocystis jirovecii	mtSSU rRNA gene