Visible and rapid detection of feline chaphamaparvovirus using multienzyme isothermal rapid amplification and lateral flow dipstick assay
Journal：Frontiers in Cellular and Infection Microbiology
Impact Factor：4.6
Feline chaphamaparvovirus (FeChPV) is a novel parvovirus, first detected in diarrheic cats in Canada in 2019. Its pathogenicity and molecular characteristics remain unclear, and it exhibits host and genetic diversity, requiring further epidemiological investigation. Currently, there is no standardized method for FeChPV detection.
The multienzyme isothermal rapid amplification-lateral flow dipstick (MIRA-LFD) method has been successfully used for detecting various viruses, offering advantages such as rapidity, simplicity, and no need for precision instruments. The research team established a MIRA-LFD method suitable for FeChPV to address challenges in grassroots clinical detection. Without relying on precision instruments, the MIRA-LFD method completed FeChPV detection at 37°C within 20 minutes and showed no cross-reactivity with other viruses. The detection limit was 32.3 copies/μL, 10 times higher than the PCR method. Furthermore, the MIRA-LFD method detected 29 FeChPV-positive samples among 417 diarrheic cats, with a positivity rate slightly higher than that of the nested PCR method. These results indicate that the established MIRA-LFD method for detecting FeChPV is an efficient, economical, reliable, and simple method, contributing to the early prevention and control of FeChPV infection.
1.Experimental Methods
Clinical Samples: Rectal swab samples used in this study were collected from 632 cats (including 417 with diarrhea and 115 healthy cats) and 474 dogs (including 342 with diarrhea and 132 healthy dogs) at veterinary clinics in Guangdong, Henan, Anhui, Zhejiang provinces and the Inner Mongolia Autonomous Region between 2022 and 2024.
Nucleic Acid Amplification: Extracted DNA nucleic acid was added to the MIRA system. Recombinase and primers formed a complex; with the help of auxiliary proteins and single-strand binding proteins, they invaded the double-stranded DNA template. Primers bound to homologous complementary regions to form a D-loop region, while the recombinase disassembled from the complex. Polymerase bound to the 3' end of the primers and initiated DNA synthesis, exponentially amplifying the target region on the template. This process cycled rapidly and efficiently, thereby achieving ultra-fast amplification of the target fragment in just 15 minutes.
Test Strip Color Development: The nucleic acid amplification product was diluted 1:10, applied to the test strip, and results were read after 5 minutes.
The entire process from nucleic acid amplification to test strip color development took only 20 minutes.
Amp-future Biological products were used in the experiment：
In the study, four designed sets of specific primer-probe combinations were screened. Both agarose gel electrophoresis and nucleic acid test strips were used for result presentation. All four primer-probe combinations could detect the FeChPV virus. According to the agarose gel electrophoresis results, the FeChPV-4 combination showed the brightest target band, so this combination was selected for subsequent testing.
According to the agarose gel electrophoresis results, amplification for 5, 10, 15, and 20 minutes all yielded the target band. The target band intensity peaked at 15 minutes of amplification, establishing 15 minutes as the optimal reaction time for this assay. Amplification at 36°C, 37°C, 38°C, 39°C, and 40°C all produced the target band, with the brightest band observed at 39°C, establishing 39°C as the optimal reaction temperature for this assay.
(Figure A: 1: Amplification 5min; 2: Amplification 10min; 3: Amplification 15min; 4: Amplification 20min) (Figure B: 1: Amplification 36°C; 2: Amplification 37°C; 3: Amplification 38°C; 4: Amplification 39°C; 5: Amplification 40°C)
2.Detection Performance
Sensitivity:
(Figure A: MIRA results presented using agarose gel electrophoresis)
(Figure B: Nested PCR results presented using agarose gel electrophoresis)
(Figure C: MIRA results presented using nucleic acid test strips)
Samples were subjected to 10-fold serial dilutions starting from 3.23×10⁶ copies/μL. Nested PCR with agarose gel electrophoresis could stably detect 3.23×10² copies/μL; MIRA with agarose gel electrophoresis and MIRA with nucleic acid test strips could both stably detect 3.23×10¹ copies/μL.
The MIRA method showed consistent sensitivity using two different result presentation formats, and the sensitivity of the MIRA method was superior to that of nested PCR.
Specificity：
(1: FeChPV, feline chaphamaparvovirus; 2: FPV, feline panleukopenia virus; 3: FeAstV, feline astrovirus; 4: FBoV, feline bocavirus; 5: CachaV, canine chaparvovirus; 6: negative control)
The newly established MIRA-LFD method detected only FeChPV and showed no cross-reactivity with other reference viruses, indicating good specificity.
3.Study Summary
This study established a MIRA-LFD method for detecting FeChPV without the need for high-precision instruments. As an efficient, economical, and reliable detection method with high sensitivity and specificity, the MIRA-LFD method is more suitable for clinical detection, providing technical support for the early prevention and control of FeChPV infection.
4.AMP-FUTURE BIOTECH
MIRA multienzyme isothermal rapid nucleic acid amplification is a technology that truly enables on-site rapid detection.
MIRA technology is based on the in vivo gene recombination repair mechanism, relying on the synergistic action of multiple functional proteins at room temperature to achieve rapid (5-20 min), sensitive, specific, and safe nucleic acid amplification at room temperature (25°C–45°C). Due to the isothermal nature of MIRA technology, it has low equipment requirements and simple operation. Through miniaturized portable devices, it can be applied in frontline testing across diverse application scenarios, bringing rapid and accurate nucleic acid testing to settings such as POCT, home testing, agricultural fields, and on-site law enforcement. Based on MIRA technology, Amp-future Biotech has developed a series of on-site rapid testing products, including rapid nucleic acid release agents, isothermal rapid nucleic acid amplification reagents, colloidal gold test strips, and miniaturized portable equipment, creating an overall solution for on-site rapid molecular testing. Furthermore, Amp-future's MIRA technology offers application advantages when combined with CRISPR technology, NGS, SNP, microfluidics, and more.
In addition to the series of on-site rapid testing products shown above, Amp-future Biotech also provides:
Research Reagents: Providing reagent raw materials and technical support for basic scientific research, as well as project literature references.
OEM Production: Capable of independently producing high-purity, high-activity finished products throughout the entire process, achieving industrial-scale production.
Project Commissioned Development: Sequence information and sequence alignment analysis results, project primer-probe design and synthesis, project plasmid synthesis and samples, project reports and results, etc.
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