In recent years,real-time fluorescence quantitative polymerase chain reaction(qPCR)technology has become an essential tool for molecular diagnosis,pathogen detection,and gene expression analysis,thanks to its high sensitivity,speed,and real-time quantification capabilities.In 2022,the global market size of nucleic acid testing-related products and services,including instruments,reagents,consumables,and after-sales service support,reached 7.3 billion US dollars,with PCR-based technologies accounting for 66.7%of the market share and exhibiting a consistent growth trend.Although qPCR technology has been widely applied across multiple fields,the preclinical development of diagnostic kits—a process that includes primer design and reaction system optimization—still faces such issues as unclear procedures,non-standardized methods,and inconsistent evaluation criteria.Herein,we reviewed the guidelines,key resources,and standardized processes of qPCR assay reagent development,aiming to provide theoretical support for improving the efficiency and quality control of assay reagent development,and to discuss future directions for the optimizing and improving qPCR technology in the context of artificial intelligence.

It is unclear whether forearm and crural muscle fibers extend distally across the wrist and ankle joints, respectively.We hypothesized, in late-term fetuses, an over-production of muscle bellies extending over the joint. Muscle fibers in histological sections from unilateral wrists and ankles of 16 late-term fetuses (30–40 weeks) were examined and compared with 15 adult cadavers. Muscle fibers of the flexor digitorum profundus (FDP) and flexor digitorum superficialis (FDS) in fetuses, especially muscle bellies to the third and fourth fingers, were found to extend far distally beyond the radiocarpal joint.The extensor digitorum and extensor pollicis longus on the extensor side of the wrist were found to carry distally-extending muscle fibers, but these fibers did not extend beyond the distal end of the radius. In the ankle, most muscle bundles in the flexor hallucis longus (FHL), fibularis brevis (FB) and extensor digitorum longus extended distally beyond the talocrural joint, with most FB muscle fibers reaching the level of the talocalcaneal joint. In adult cadavers, muscle fibers of the FDP and FHL did not reach the levels of the radiocarpal and talocrural joints, respectively, whereas the FB muscle belly always reached the talocalcaneal joint. Similarly, some of the FDS reached the level of the radiocarpal joint. Generally, infants’ movements at the wrist and ankle could result in friction injury to over-extended muscle. However, the calcaneal and FDP tendons might protect the FB and FDS tendons, respectively, from friction stress.