ABSTRACT
Conventional PCR methods can detect only a few targets simultaneously and do not fulfill most clinical requirements, especially those for detecting plasma circulating DNA. By designing characteristic universal fluorescent probes, combining multiplex PCR with the invasive reaction, and analyzing the resulting differences in the melting curves formed by extension with double-stranded probes, we developed a new method to distinguish between three mutations in the same fluorescent channel and nine mutations in three fluorescent channels in a single tube. After optimization, this method was used to distinguish between 27 mutations using only three reactions, and mutations representing as low as 0.2%-0.5% of DNA could be detected, even when up to nine mutations were present at the same time. Testing of nine clinical samples, including three L858R-positive, four 19 del-positive, and two L861Q-positive samples, showed consistent results with digital PCR tests. Compared with the conventional PCR method, our method expands the capabilities of fluorescence detection by achieving multiplex detection in a single-tube, thereby providing a simple, low-cost tool for clinical applications.
ABSTRACT
BACKGROUND:Dural repair materials in current application mainly include autologous tissue repair material, alograft material, heterogeneous biological material and synthetic material, most of which are imported products with expensive price. OBJECTIVE: To evaluate safety and efficacy of a new biological type dura mater patch made in China based on animal experiments. METHODS:Bilateral dura mater defect models were established in 24 healthy domestic dogs: on the left side of the implant model, a new type biological dura patch was transplanted as experimental group; on the right side, another brand artificial dura patch that was on sale was transplanted as control group. After 1, 3, 6 and 12 months of implantation, we compared degradation, angiogenesis, growth and surrounding tissue reaction of dural substitutes of the experimental group and control group by hematoxylin-eosin staining, detected residual dose of epoxy-cross-linked agent in dogs’ blood and cerebrospinal fluid by fluorescence spectrophotometry. RESULTS AND CONCLUSION: During 1-12 months of implantation, al dogs grew wel and no infection or motor disorder was observed. Pathological examination showed that dura substitutes of the experimental group and control group had good biocompatibility, no or slightly inflammatory response. After 6 months of implantation, the surface of the new biological dural substitute (experimental group) was degraded and became a transit-state biomaterial with surrounding tissue, but the control group materials showed no degradation. After 12 months of implantation, the dura patch in the experimental group degraded nearly 50%, which appeared with neovascularization; while, the dura patch in the control group degraded 30%, and neovascularization was observed in only a smal amount of samples. Epoxy compounds of cross-linked agent were not detected in dogs’ blood and cerebrospinal fluid after 1, 3, 7 and 14 postoperative days. These findings show that this new type of biological dural substitute is a safe and effective dural repair material.
