Inhibition of Carbohydrate Metabolism Potentiated by the Therapeutic Effects of Oxidative Phosphorylation Inhibitors in Colon Cancer Cells.

Cancer cells undergo a significant level of "metabolic reprogramming" or "remodeling" to ensure an adequate supply of ATP and "building blocks" for cell survival and to facilitate accelerated proliferation. Cancer cells preferentially use glycolysis for ATP production (the Warburg effect); however, cancer cells, including colorectal cancer (CRC) cells, also depend on oxidative phosphorylation (OXPHOS) for ATP production, a finding that suggests that both glycolysis and OXPHOS play significant roles in facilitating cancer progression and proliferation. Our prior studies identified a semisynthetic isoflavonoid, DBI-1, that served as an AMPK activator targeting mitochondrial complex I. Furthermore, DBI-1 and a glucose transporter 1 (GLUT1) inhibitor, BAY-876, synergistically inhibited CRC cell growth in vitro and in vivo. We now report a study of the structure-activity relationships (SARs) in the isoflavonoid family in which we identified a new DBI-1 analog, namely, DBI-2, with promising properties. Here, we aimed to explore the antitumor mechanisms of DBIs and to develop new combination strategies by targeting both glycolysis and OXPHOS. We identified DBI-2 as a novel AMPK activator using an AMPK phosphorylation assay as a readout. DBI-2 inhibited mitochondrial complex I in the Seahorse assays. We performed proliferation and Western blotting assays and conducted studies of apoptosis, necrosis, and autophagy to corroborate the synergistic effects of DBI-2 and BAY-876 on CRC cells in vitro. We hypothesized that restricting the carbohydrate uptake with a KD would mimic the effects of GLUT1 inhibitors, and we found that a ketogenic diet significantly enhanced the therapeutic efficacy of DBI-2 in CRC xenograft mouse models, an outcome that suggested a potentially new approach for combination cancer therapy.

Double-layered N-S1 protein nanoparticle immunization elicits robust cellular immune and broad antibody responses against SARS-CoV-2.

BACKGROUND: The COVID-19 pandemic is a persistent global threat to public health. As for the emerging variants of SARS-CoV-2, it is necessary to develop vaccines that can induce broader immune responses, particularly vaccines with weak cellular immunity. METHODS: In this study, we generated a double-layered N-S1 protein nanoparticle (N-S1 PNp) that was formed by desolvating N protein into a protein nanoparticle as the core and crosslinking S1 protein onto the core surface against SARS-CoV-2. RESULTS: Vaccination with N-S1 PNp elicited robust humoral and vigorous cellular immune responses specific to SARS-CoV-2 in mice. Compared to soluble protein groups, the N-S1 PNp induced a higher level of humoral response, as evidenced by the ability of S1-specific antibodies to block hACE2 receptor binding and neutralize pseudovirus. Critically, N-S1 PNp induced Th1-biased, long-lasting, and cross-neutralizing antibodies, which neutralized the variants of SARS-CoV-2 with minimal loss of activity. N-S1 PNp induced strong responses of CD4+ and CD8+ T cells, mDCs, Tfh cells, and GCs B cells in spleens. CONCLUSIONS: These results demonstrate that N-S1 PNp vaccination is a practical approach for promoting protection, which has the potential to counteract the waning immune responses against SARS-CoV-2 variants and confer broad efficacy against future new variants. This study provides a new idea for the design of next-generation SARS-CoV-2 vaccines based on the B and T cells response coordination.

Enhancing humoral and mucosal immune response of PED vaccine candidate by fusing S1 protein to nanoparticle multimerization.

Porcine epidemic diarrhea virus (PEDV) is a highly infectious pathogen with a high mortality rate, which poses a serious threat to newborn piglets. A rapid, safe and effective vaccine is necessary for protecting pigs from PED infection. Nanoparticles have become molecular scaffolds for displaying soluble antigens due to their unique physical and chemical properties. Here, a vaccine candidate was based on the display of PEDV S1 protein on a mi3 nanoparticle platform using SpyTag/SpyCatcher technology. The size, zeta potential and microstructure of the S1-mi3 NPs were investigated, and their effects on the uptake of antigen-presenting cells (APCs) and maturation of dendritic cells (DCs) were analyzed. Mice were immunized via muscular and intranasal administrations, and the levels of humoral, cellular and mucosal immune responses were analyzed. As a result, S1 proteins were surface-displayed on NPs successfully, which self-assembled into nanoparticles composed of 60 subunits and showed superior safety and stability. In addition, mi3 NPs promoted antigen internalization and dendritic cell (DCs) maturation. In the mouse model, S1-mi3 NPs significantly increased the PEDV-specific antibody including serum IgG, secretory IgA (SIgA) and neutralizing antibodies (NAb). Furthermore, S1-mi3 NPs elicited more CD3+CD4+ and CD3+CD8+ T cell and cellular immune-related cytokines (IFN-γ and IL-4) compared to monomeric S1. In particular, it can induce an effective germinal center-specific (GC) B cell response, which is closely related to the production of neutralizing antibodies. Overall, S1-mi3 NPs are a promising subunit vaccine candidate against PEDV, and this self-assembly NPs also provide an attractive platform for improving vaccine efficacy against emerging pathogens.

Screening, Expression and Identification of Nanobody Against Monkeypox Virus A35R.

Introduction: The monkeypox (Mpox) virus epidemic presents a significant risk to global public health security. A35R, a crucial constituent of EEV, plays a pivotal role in virus transmission, serves as a vital target for vaccine development, and has potential for serological detection. Currently, there is a dearth of research on nanobodies targeting A35R. The purpose of this study is to identify specific nanobodies target A35R, so as to provide new antibody candidates for Mpox vaccine development and diagnostic kit development. Methods: Three nanobodies specific to the monkeypox virus protein A35R were screened from a naïve phage display library. After four rounds of panning, positive phage clones were identified by enzyme-linked immunosorbent assay (ELISA). Further, the nanobody fusion protein was constructed in pNFCG1-IgG1-Fc vector and expressed in HEK293F cells and purified by affinity chromatography. The specificity and affinity of the nanobodies were identified by ELISA. The binding kinetics of the VHH antibody to A35R were assessed via employment of a bio-layer interferometry (BLI) apparatus, thereby determining the nanobodies affinity. Results: The three purified nanobodies showed specific high-affinity binding MPXV A35R, of them, VHH-1 had the best antigen binding affinity (EC50 = 0.010 ug/mL). In addition, VHH-1 on Protein A biosensor can bind Mpox virus A35R, with an affinity constant of 54 nM as determined in BLI assay. Conclusion: In sum, we has obtained three nanobody strains against Mpox virus A35R with significant affinity and specificity, therefore laying an essential foundation for further research as well as the applications of diagnostic and therapeutic tools of Mpox virus.

Fusion imaging versus ultrasound-guided percutaneous thermal ablation of liver cancer: a meta-analysis.

BACKGROUND: Ultrasound-guided percutaneous thermal ablation has become an alternative treatment for small hepatocellular carcinoma (HCC). Recent evidence suggests that fusion imaging (FI) may improve the feasibility and efficacy of thermal ablation for HCC, while the clinical evidence remains limited. PURPOSE: To compare FI versus ultrasound-guided thermal ablation for HCC. MATERIAL AND METHODS: Relevant cohort or randomized controlled trials were found by searching Medline, Web of Science, Cochrane Library, and Embase. The pooling of results was performed using a random-effects model incorporating heterogeneity. RESULTS: In this meta-analysis, 15 studies involving 1472 patients (1831 tumors) for FI-guided ablation and 1380 patients (1864 tumors) for ultrasound-guided ablation were included. Pooled results showed that compared to conventional HCC ablation guided by ultrasound, the FI-guided procedure showed a similar technique efficacy rate (risk ratio [RR] = 1.01, 95% confidence interval [CI] = 1.00-1.02, P = 0.25; I2 = 30%). However, FI-guided tumor ablation was associated with a lower incidence of overall complications (RR = 0.70, 95% CI = 0.50-0.97, P = 0.03; I2 = 0%). Moreover, patients receiving FI-guided tumor ablation had a lower risk of local tumor progression during follow-up than those with ultrasound-guided ablation (RR = 0.61, 95% CI = 0.47-0.78, P < 0.001; I2 = 13%). Subgroup analysis according to FI strategy, imaging techniques in controls, and tumor diameter showed consistent results (p for subgroup difference all >0.05). CONCLUSION: FI-guided thermal ablation may be more effective and safer than ultrasound-guided ablation for patients with HCC.

Development and characterization of monoclonal antibody against the critical loop structure of african swine fever virus P72 protein.

African swine fever (ASF) is a highly infectious and lethal viral disease caused by the African swine fever virus (ASFV). The four prominent loop structures on the surface of the primary structural protein P72 are considered to be key protective epitopes. In this study, the four critical loops (ER1-4) of the ASFV p72 protein were individually fused to hepatitis B virus core particles (HBc) and self-assembled into nanoparticles to preserve the natural conformation of the loop structure and enhance its immunogenicity. Then, four recombinant proteins were obtained in E. coli expression system and monoclonal antibodies (mAbs) were developed and characterized. All 10 mAbs obtained were able to react with P72 protein and ASFV with potencies up to 1:204 800. Amino acids 250-274, 279-299 and 507-517 of the P72 protein were identified as linear epitopes and highly conserved. The mAb 4G8 showed the highest inhibition rate of 84% against ASFV positive sera. Importantly, neutralization experiments illustrated that mAb 4G8 has a 67% inhibition rate, indicating that its corresponding epitopes are potential candidates for ASFV vaccine. In conclusion, highly immunogenic nanoparticles of the ASFV P72 key loop were constructed to induce the production of highly effective mAbs and clarify their epitope information for the diagnosis and prevention of ASFV.

Spy&IAC enables specific capture of SpyTagged proteins for rapid assembly of plug-and-display nanoparticle vaccines.

From modular vaccine production to protein assembly on nanoparticles, the SpyCatcher/SpyTag system provides a convenient plug-and-display procedure. Here, we established a general-purpose immunoaffinity chromatography (IAC) method for SpyTagged proteins (Spy&IAC). SpyTags are displayed on the surface of nanoparticles to induce high-affinity monoclonal antibodies, allowing the specific capture of the target protein. Taking the key core antigenic regions of two coronaviruses that are currently more threatened in the field of human and animal diseases, the nucleocapsid (N) protein of SARS-CoV-2 and the COE protein of porcine epidemic diarrhea virus (PEDV) as model proteins, a purification model with SpyTag at the N-terminal or C-terminal expressed in E. coli or mammalian cells was constructed. After the efficient elution of Spy&IAC, the final yield of several proteins is about 3.5-15 mg/L culture, and the protein purity is above 90 %. Purification also preserves the assembly function and immunogenicity of the protein to support subsequent modular assembly and immunization programs. This strategy provides a general tool for the efficient purification of SpyTagged proteins from different expression sources and different tag positions, enabling the production of modular vaccines at lower cost and in a shorter time, which will prepare the public health field for potential pandemic threats.

Development and characterization of monoclonal antibodies against the extracellular domain of African swine fever virus structural protein, CD2v.

African swine fever virus (ASFV), a DNA double-stranded virus with high infectivity and mortality, causing a devastating blow to the pig industry and the world economy. The CD2v protein is an essential immunoprotective protein of ASFV. In this study, we expressed the extracellular region of the CD2v protein in the 293F expression system to achieve proper glycosylation. Monoclonal antibodies (mAbs) were prepared by immunizing mice with the recombinant CD2v protein. Eventually, four mAbs that target the extracellular region of the ASFV CD2v protein were obtained. All four mAbs responded well to the ASFV HLJ/18 strain and recognized the same linear epitope, 154SILE157. The specific shortest amino acid sequence of this epitope has been accurately identified for the first time. Meaningfully, the 154SILE157 epitope was highly conformed in the ASFV Chinese epidemic strain and Georgia2008/1 strains according to the analysis of the conservation and have a fair protective effect. These findings contribute to further understanding of the protein function of CD2v and provide potential support for the development of diagnostic tools and vaccines for ASFV.

Ce6-C6-TPZ co-loaded albumin nanoparticles for synergistic combined PDT-chemotherapy of cancer.

Photodynamic therapy (PDT), as an essential tumor treatment method, has received great attention; however, there are still some challenges such as hydrophobicity of most of the photosensitizers, safety of in vivo transport, and characteristics of oxygen consumption. Herein, we used albumin as the nanocarrier for the loading of Chlorin e6 (Ce6) photosensitizer. In the meantime, tirapazaming (TPZ) was co-loaded onto the nanocomposite, which could be activated by hypoxia caused by PDT for enhanced therapy. Considering the over irradiation problem, a strategy for measuring PDT degree by ratio fluorescence was utilized. The PDT monitoring design relies on ratio emissions of C6 (Coumarin 6) and Ce6 molecules since the red emission of Ce6 is dependent on the PDT capability. Based on the characterization of the albumin nanocomposites, we further explored the combined therapy effect at both the in vitro and in vivo levels and attained the corresponding results.

Noninvasive temperature monitoring for dual-modal tumor therapy based on lanthanide-doped up-conversion nanocomposites.

Accurate treatment of photothermal therapy (PTT) is crucial to avoid the unnecessary injury of normal cells and tissues. Therefore the real-time temperature monitoring in the PTT process has drawn more and more attention in recent years. Herein, we designed and prepared one kind of lanthanide (Ln3+)-doped up-conversion nanocomposites with multi-functions, which can not only provide temperature feedback in PTT process, but also play the photodynamic therapy (PDT) function for the synergistic effect of tumor therapy. Based on NaYF4:Yb, Er up-conversion nanoparticles (UCNPs), mesoporous SiO2 was modified on the surface combined with photosensitizer Chlorin e6 (Ce6) molecules, which could be excited by red emission of Er3+ under the 980 nm laser. Cit-CuS NPs were further linked on the surface of the composite served as photothermal conversion agent, therefore, the temperature of the PTT site can be monitored by recording the ratio of I525/I545 of green emissions, especially within the physiological range. Based on the guidance obtained from spectral experiments, we further investigated the dual-modal therapy effect both in vitro and in vivo, respectively, and acquired decent results.

Nanoparticles having amphiphilic silane containing Chlorin e6 with strong anti-biofilm activity against periodontitis-related pathogens.

OBJECTIVES: The objectives of this study were to: (1) develop the multifunctional nanoparticles containing Chlorin e6 (Ce6), Coumarin 6 (C6) and Fe3O4 nanoparticles (NPs); and (2) investigate the inhibitory effects of the nanoparticles via antibacterial photodynamic therapy (aPDT) against three species of periodontitis-related pathogens for the first time. MATERIALS AND METHODS: Ce6 and C6 were co-loaded into the Fe3O4-silane core-shell structure to form multifunctional nanoparticles (denoted "Fe3O4-silane@Ce6/C6 MNPs"). The physical and chemical properties of nanoparticles were characterized. Biofilm properties of Streptococcus sanguinis, Porphyromonas gingivalis and Fusobacterium nucleatum were tested. Colony-forming units (CFU), live/dead assay, and metabolic activity of biofilms were determined to evaluate the aPDT function mediated by the Fe3O4-silane@Ce6/C6 MNPs. Fluorescence imaging and the targeted antibacterial effects were also investigated. RESULTS: Fe3O4-silane@Ce6/C6 MNPs showed superparamagnetic properties, chemical stability and water-solubility, with no cytotoxicity. Fe3O4 NPs did not compromise the emission peaks of C6 and Ce6. The Fe3O4-silane@Ce6/C6-mediated aPDT had much greater reduction in biofilms than the control groups (p < 0.05). Biofilm CFU was reduced by about 4-5 orders of magnitude via Fe3O4-silane@Ce6/C6-mediated aPDT. The co-loading of Ce6 and C6 enabled the real-time aPDT monitoring by ratio emissions with the same wavelength. Fe3O4 with magnetic field enabled the targeting of infection sites by killing bacteria via magnetic field. CONCLUSION: The multifunctional nanoparticles exerted strong anti-biofilm activity against periodontitis-related pathogens, with excellent biocompatibility, real-time monitoring, and magnetically-targeting capacities. The multifunctional nanoparticles have great potential in antibacterial applications to inhibit the occurrence and progression of periodontitis.

Luminescence carbon dot-based nanofibers for a water-insoluble drug release system and their monitoring of drug release.

Drug release systems with fluorescence detection have emerged as a potential application for the biological area of diagnosis and therapy. Carbon dots (CDs) are a promising fluorescence probe for application in a drug release system due to their excellent biocompatibility, low toxicity, chemical inertness, and non-blinking fluorescence emission. Herein, we developed a composite nanocarrier based on fluorescent CDs and polyvinylpyrrolidone (PVP) through an electrospinning technology. The as-prepared PVP/CD-ketoprofen (PVP/CD-KET) composite nanofiber presents bright blue-light fluorescence with a photoluminescence quantum yield (QY) of 65.7% and was utilized for loading a water-insoluble drug and moreover for monitoring the drug release process. In vitro tests indicate that the photoluminescence emission intensity of the CDs and the cumulative amount of KET released from the PVP/CD-KET composite nanofiber gradually increase with the release time. Furthermore, the emission intensity of the CDs as a function of the cumulative released amount of KET can be summarized by a power function. The correlation between the emission intensity of CDs and drug release amount can be potentially used to monitor the drug release process.

Ratiometric photoluminescence sensing based on Ti3C2 MXene quantum dots as an intracellular pH sensor.

Intracellular pH sensing is of importance and can be used as an indicator for monitoring the evolution of various diseases and the health of cells. Here, we developed a new class of surface-functionalized MXene quantum dots (QDs), Ti3C2, by the sonication cutting and hydrothermal approach and further explored their intracellular pH sensing. The functionalized Ti3C2 QDs exhibit bright excitation-dependent blue photoluminescence (PL) originating from the size effect and surface defects. Meanwhile, Ti3C2 QDs demonstrate a high PL response induced by the deprotonation of the surface defects. Furthermore, combining the highly pH sensitive Ti3C2 QDs with the pH insensitive [Ru(dpp)3]Cl2, we developed a ratiometric pH sensor to quantitatively monitor the intracellular pH values. These novel MXene quantum dots can serve as a promising platform for developing practical fluorescent nanosensors.

Three-Dimensional Inverse Opal Photonic Crystal Substrates toward Efficient Capture of Circulating Tumor Cells.

Artificial fractal structures have attracted considerable scientific interest in circulating tumor cells (CTCs) detection and capture, which plays a pivotal role in the diagnosis and prognosis of cancer. Herein, we designed a bionic TiO2 inverse opal photonic crystal (IOPC) structure for highly efficient immunocapture of CTCs by combination of a magnetic Fe3O4@C6@silane nanoparticles with anti-EpCAM (antiepithelial cell adhesion molecule) and microchannel structure. Porous structure and dimension of IOPC TiO2 can be precisely controlled for mimicking cellular components, and anti-EpCAM antibody was further modified on IOPC interface by conjugating with polydopamine (PDA). The improvement of CTCs capture efficiency reaches a surprising factor of 20 for the IOPC interface compared to that on flat glass, suggesting that the IOPCs are responsible for the dramatic enhancement of the capture efficiency of MCF-7 cells. IOPC substrate with pore size of 415 nm leads to the optimal CTCs capture efficiency of 92% with 1 mL/h. Besides the cell affinity, IOPCs also have the advantage of light scattering property which can enhance the excitation and emission light of fluorescence labels, facilitating the real-time monitoring of CTCs capture. The IOPC-based platform demonstrates excellent performance in CTCs capture, which will take an important step toward specific recognition of disease-related rare cells.

High purity microfluidic sorting and in situ inactivation of circulating tumor cells based on multifunctional magnetic composites.

Detection and isolation of circulating tumor cells (CTCs) play a pivotal role in the diagnosis and prognosis of cancer, while the high capture efficiency and purity of CTCs are difficult to achieve simultaneously among the various isolation methods. In this work, we designed an inverted microchip integrating silicon nanowires (SiNWs) and multifunctional magnetic nanocomposites (Fe3O4@C6/Ce6@silane, Coumarin 6 (C6), Chlorin e6 (Ce6)) for enhanced capture efficiency and purity of CTCs. The Fe3O4@C6/Ce6@silane conjugated with antibody can label the CTCs and pull them to the upside SiNWs capture surface by the upward magnetic field with high purity. This inverted structure was also featured with real-time detection and photodynamic therapy (PDT) of CTCs with the confocal laser scanning microscope (CLSM). The results indicate the important role of the composites labels and the magnetic field, which greatly improves the capture purity of the CTCs to 90%. Meanwhile, capture efficiency of CTCs achieve to 90.3% in culture medium and 82% in blood with 2 mL/h flow rate, respectively. Based on the structure of the device and composites, the captured CTCs could be directly inactivated by the in situ photodynamic therapy in the capture process which holds positive impact to block cancer spread.

Amphiphilic Silane Modified Multifunctional Nanoparticles for Magnetically Targeted Photodynamic Therapy.

Efficient targeting is a major challenge in practical photodynamic therapy (PDT). Though the "enhanced permeability and retention" (EPR) effect is a widely used tumor targeting method, magnetic targeting strategy is more promising considering the issue of high targeting efficiency and reducing concentration-dependent toxicity. Herein, magnetic targeting and highly effective Fe3O4@Ce6/C6@silane NPs are reported as a class of precisely controlled photosensitizers (PS) for PDT. On the basis of the amphiphilic silane encapsulation, PS chlorin e6 (Ce6) and Coumarin 6 (C6) as well as Fe3O4 NPs were coloaded into the inside hydrophobic environment of amphiphilic silane, forming a theranostic agent for dual-mode imaging guided and magnetic targeting enhanced in vivo PDT agent. To solve the problem of over-irradiation, the coloaded design of C6 and Ce6 molecules can afford the real time PDT monitoring by ratio emissions with same excitation wavelength. When Fe3O4@Ce6/C6@silane and Ce6/C6@silane NPs are compared in in vitro and in vivo experiments, the introduction of Fe3O4 in the composite does not affect the PDT efficiency, whereas, in contrast, it brings MRI imaging and magnetic targeting functions. Fe3O4@Ce6/C6@silane injection followed with magnetic field (MF) and light irradiation is important in generating an effective PDT process, showing great potential in tumor therapy.

A dendrimer-based highly sensitive and selective fluorescence-quenching sensor for Fe(3+) both in solution and as film.

A novel fluorescent dendrimer PYTPAG2, with pyrene as the interior core and triphenylamine (TPA) as the exterior periphery, is studied as a fluorescence-quenching sensor for iron (ш) ions (Fe(3+)), both in solution and as a film. This dendrimer-based sensor possesses preferential detection of Fe(3+) by a very strong fluorescence quenching not found for other metal ions. The fluorescent detection limits of this PYTPAG2 sensor for Fe(3+) in solution and thin-film are 6.5×10(-7)M and 5.0×10(-7)M, respectively. The possible mechanism of this process is explained by the complexation between the peripheral TPA units of PYTPAG2 and Fe(3+) ions, which may disrupt the fluorescence resonance energy transfer (FRET) from the TPA groups to the pyrene core (intramolecular of PYTPAG2) and results in the fluorescence quenching. Moreover, this striking performance could not be disturbed by pH, the interference with other metal ions, counter anions, or surrounding environment. In addition, biological fluorescence imaging studies of Fe(3+) in living roundworms demonstrate its valuable practical application.

[Gene mapping of developmental dysplasia of the hip in chromosome 17q21 region].

OBJECTIVE: To map the susceptibility gene of developmental dysplasia of the hip(DDH) in chromosome 17q21 region. METHODS: According to the number of alleles (≥ 5), heterozygosity (≥ 0.70) and polymorphic information content (PIC≥ 0.5), 11 STR markers in the 17q21 region were chosen for transmission disequilibrium test (TDT). STR markers were amplified by PCR and genotypes were analyzed by capillary electrophoresis in 103 trio families. TDT was used to locate the susceptibility gene in 17q21 region. RESULTS: Because of a low genetic polymorphism, D17S810 and D17S931 loci were removed from the TDT. Transmission disequilibrium was detected at D17S855, D17S858, D17S806, D17S1877, D17S941, D17S752 and D17S790, which overlapped 11.7 cM in 17q21. However, no transmission disequilibrium was found at D17S1787 and D17S787. Thus, the susceptibility gene for DDH was located in the chromosome region between D17S855 and D17S790. CONCLUSION: The susceptibility gene for DDH is narrowed to an 11.7 cM region of 17q21.31-17q22, between STR loci D17S855 and D17S790.

[A new method for SNP typing based on allele specific PCR].

OBJECTIVE: To establish a new method of SNP typing. METHODS: Based on the principle of allele specific PCR and capillary electrophoresis technique, 11 diallelic SNP loci were selected and two forward primers with different length were designed for each SNP, with their 3' ends matched to the two alleles, respectively. An artificially mismatched base was also introduced into the third or fourth base in the 3' end area of the two forward primers in order to enhance the specificity of amplification. A common reverse primer was designed 100-300 bp away from the forward primers, and labeled with fluorescence. The PCR products were separated and analyzed by ABI Prism 310 Genetic Analyzer after all of the 11 SNPs were multiply amplified. RESULTS: A single product peak was observed while the SNP was homozygous, and two product peaks with different height were observed while the SNP was heterozygous. The length of PCR products was different with the different SNPs. According to the length of the products and the number of the product peaks, the genotypes of the 11 SNPs can simultaneously be analyzed, and the results were in accordance with the direct sequencing. CONCLUSION: Fragment length discrepant allele specific fluorescence labeled multi-PCR (FLDASFLM-PCR) is a simple, rapid and efficient new method for SNP typing.