Application of recombinase polymerase amplification with CRISPR/Cas12a and multienzyme isothermal rapid amplification with lateral flow dipstick assay for Bactrocera correcta.

BACKGROUND: Bactrocera correcta is a quarantine pest that negatively impacts the fruit and vegetable industry. Differentiating B. correcta from similar species, especially in non-adult stages, remains challenging. Rapid molecular identification techniques, such as recombinase polymerase amplification (RPA) combined with CRISPR/Cas12a and multienzyme isothermal rapid amplification with lateral flow dipstick (MIRA-LFD), play a crucial role in early monitoring and safeguarding agricultural production. Our study introduces two methods for the rapid visual identification of B. correcta. RESULTS: Bactrocera correcta specific RPA primers, CRISPR RNA (crRNA), and the LFD probe were designed based on the cox1 genes. The RPA reaction conditions were optimized (at 37 °C for 8 min) for effective template DNA amplification. Two nucleic acid detection methods were established to visualize RPA. In the RPA-CRISPR/Cas12a system, the optimal LbCas12a/crRNA concentration ratio was 200:400 nmol L-1. Successful amplification was determined by the presence or absence of green fluorescence following 15 min incubation at 37 °C. The MIRA-LFD system achieved precise identification of the target species within 4 min at 37 °C. Both methods exhibited high specificity and sensitivity, allowing for detection from 1.0 × 10-1 ng µL-1 of DNA. Combined with rapid DNA extraction, rapid identification of individual B. correcta at different developmental stages was achieved, enhancing the practicality and convenience of the established methods. CONCLUSION: Our research findings demonstrate that both the RPA-CRISPR/Cas12a and MIRA-LFD methods for B. correcta detection was accurate and rapid (within 30 min and 10 min, respectively), at 37 °C. Our methods do not rely on expensive equipment, thus possess high practical value, providing improved identification solutions for port quarantine pests and field applications. © 2024 Society of Chemical Industry.

Mitochondrial dysfunction precedes hippocampal IL-1ß transcription and cognitive impairments after low-dose lipopolysaccharide injection in aged mice.

Acute cognitive impairments termed delirium often occur after inflammatory insults in elderly patients. While previous preclinical studies suggest mitochondria as a target for reducing neuroinflammation and cognitive impairments after LPS injection, fewer studies have evaluated the effects of a low-grade systemic inflammation in the aged brain. Thus, to identify the significance of mitochondrial dysfunction after a clinically relevant systemic inflammatory stimulus, we injected old-aged mice (18-20 months) with low-dose lipopolysaccharide (LPS, 0.04 mg/kg). LPS injection reduced mitochondrial respiration in the hippocampus 24 h after injection (respiratory control ratio [RCR], state3u/state4o; control = 2.82 ± 0.19, LPS = 2.57 ± 0.08). However, gene expression of the pro-inflammatory cytokine IL-1ß was increased (RT-PCR, control = 1.00 ± 0.30; LPS = 2.01 ± 0.67) at a more delayed time point, 48 h after LPS injection. Such changes were associated with cognitive impairments in the Barnes maze and fear chamber tests. Notably, young mice were unaffected by low-dose LPS, suggesting that mitochondrial dysfunction precedes neuroinflammation and cognitive decline in elderly patients following a low-grade systemic insult. Our findings highlight mitochondria as a potential therapeutic target for reducing delirium in elderly patients.