Objective Cloning-sequencing is a common method to detect the number of trinucleotide repeats.The aim of the present study is to discuss its reliability.Methods One clinically diagnosed SCA1 patient was recruited in the study.The numbers of CAG repeats in ATXN1 gene were estimated via polymerase chain reaction (PCR) and denaturing polyacrylamide gel electrophoresis (DPAGE).To verify accuracy of CAG numbers estimated, the PCR products were electrophoresed on a 2.5% agarose ge] and separated bands were excised for direct sequencing.Also, the longer separated band underwent cloning-sequencing using a TA cloning kit.Results The patient was identified as SCA1 by DPAGE.After direct sequencing, the numbers of CAG repeats were 26 and 47 in the shorter and longer bands, respectively.However, after cloning-sequencing of the longer band, there are 10 different numbers of CAG repeats, including 50, 47, 46, 41,32, 28, 27, 26, 25 and 24.Furthermore, there are other kinds of trinucleotide repeats, such as CCG, CGG, CTG, CAA and TAT scattered among the CAG repeats.Conclusions It is not reliable to identify the number of trinucleotide repeats by cloning-sequencing alone.To improve the reliability, it is better to combine cloning-sequencing with other methods.

Objective: To observe the promoting blood circulation by removing blood stasis of Lubai Capsule(LBC)(Rhizoma Phragmitis, Radix Paeoniae Alba, Radix Saposhnikoviae, Flos Schizonepetae, etc.). Methods: Acute blood stasis rat models were established with swimming in iced water and sc adrenalin in order to observe the effect of LBC on blood rheology. Mesenteric microcirculatory disturbance rat models were also established with adrenalin in order to observe the effect of LBC. Clotting time was measured in vitro with prothrombin time(PT) and kaolin partial thromboplastin time(KPTT) kit in order to observe its effects. Results: LBC could decrease the whole blood and plasma viscosity, fibrinogen, erythrocyte sedimentation and aggregation ratio of blood platelets of rats, ease the sticky condition of blood stasis rat models and prevent from forming thrombus. It could also inhibit the constraction and slowing of blood flow of thin artery, the reducing of open capillaries and change of fluid condition caused by adrenalin and improve these phenomena. PT and KPTT could be increased obviously. Conclusion: LBC can significantly promote blood circulation by removing blood stasis, because of improving blood rheology and mesenteric microcirculatory disturbance and inhibit endogenous and exogenous coagulation system.

Objective To evaluate and compare the analytical performances and application values of three nucleic acid extraction methods for quantification of plasma Epstein-Barr Virus ( EBV ) DNA. Methods It used silica membrane spin column , boiling and automated magnetic bead method to extract viral nucleic acid in parallel , and combined real-time fluorescence quantitative PCR assays for quantitative EBV-DNA quantification.The performances of three methods were determined and compared by using the third-party reference materials , and the clinical values were analyzed by pairing detecting 100 NPC patients and 100 healthy subjects in pair .Results The accuracy and imprecision of three methods were all in line with requirements , and the results of clinical samples were linearly correlated . But actually the reproducibility and intermediate imprecision of the magnetic bead method were smaller and stable than those of the spin column method and the boiling method ( all <3％);the limit of detection for the magnetic bead method was 3.334 ×101 IU/ml, better than that of spin column method (4.159 ×101 IU/ml) and boiling method (8.511 ×101 IU/ml);the linear range of the magnetic bead method was 5.4 ×101 -5.4 ×105 IU/ml, slightly wider than that of the boiling method (5.4 ×102 -5.4 ×105 IU/ml); the ability of anti -Hb interference ability of magnetic bead method is better than that of boiling method ;and the positive rate and the mean viral load of the NPC samples measured with the magnetic bead method were significantly higher (95％, 8.342 ×103 IU/ml) than those measured with the spin column method (84％, 4.707 ×103 IU/ml) and the boiling method (78％, 2.571 ×103 IU/ml) ( P all<0.05).Conclusion The automated magnetic bead nucleic acid extraction method offered better analytical performance and higher clinical value for EBV DNA quantification in plasma .