A retrospective study was conducted based on the clinical data of 42 patients of portal hypertension and splenomegaly who underwent laparoscopic splenectomy.The patients were divided into two groups including pedicle priority group and conventional group by different operative method.The operation time and intraoperative blood loss in the pedicle priority group were significantly lower than those in the conventional group (both P ＜ 0.05),and there was no statistically significant difference on the conversion rate of laparotomy,active time postoperation,exhaust time,postoperative hospitalization stay and the incidence of complications (all P ＞ 0.05).Priority processing for splenic pedicle has obvious advantages in laparoscopic splenectomy for portal hypertension and splenomegaly,and it could reduce the difficulty of operation,shorten the operation time and reduce bleeding.

Objective To analyze CT features and related factors of mediastinal lymphoma with necrosis in order to identify other mediastinal tumor with necrosis.Methods CT features were retrospectively reviewed and related factors of necrosis were analyzed in 37 cases of mediastinal lymphoma confirmed by pathology.Results 37 cases appeared as mediastinal masses,in which 25 cases were cross-regional growth of the mediastinum,1 1 cases in anterior mediastinum and 1 case in posterior mediastinum.The largest cross-sectional area of tumors was 1.6-129.6 cm2 ,in which there were 1 case (0-10)cm2 ,8 cases (10-25)cm2 ,10 cases (25-50)cm2 ,1 5 cases(50-100)cm2 and 3 cases >100 cm2 .24 cases had necrosis ,among which 23 cases were slight enhanced and 1 case was seriously enhanced.Necrosis of mediastinal lymphoma was related to the size and CT enhancement of tumor by the logistic regression analysis(P <0.05).Conclusion Mediastinal lymphoma with necrosis is common .The necrosis of mediastinal lymphoma is related to the size and CT enhancement.Slight CT enhancement is one of differential points between mediastinal lymphoma with necrosis and other mediastinal tumor.

OBJECTIVETo establish an improved, simple and convenient megaprimer PCR method for site-directed mutagenesis(SDM).

METHODSThis protocol is based on the design of two different plasmid DNA templates. Template 1 lacks the binding site for reverse flanking primer, and template 2 lacks the binding site for forward flanking primer. This modification avoids amplification of full-length wild-type sequences while two templates, the forward and reverse flanking primers exist simultaneously in one reaction tube. A megaprimer is synthesized in the first PCR reaction using template 1, forward primer and mutagenic primer. The megaprimer that needn't the cumbersome gel purification step is directly added to the second PCR reaction system. The second PCR reaction(PCR 2) containing two stages is performed using template 2, megaprimer, forward and reverse flanking primer. During the first stage of PCR 2, the megaprimer is extended to form the full-length mutation product. In the second stage of PCR 2, the extended megaprimer containing SDM residues is subsequently amplified with the two flanking primers. All of the final PCR products contained the desired mutation.

RESULTSFifteen types of rare beta-thalassemia mutations in Chinese were obtained using this method. Each of these modified fragments was separately cloned into the pGEM-T vector and sequenced. The desired mutations involved in mutagenesis amplicons were identified in all clones.

CONCLUSIONThis improved PCR-based megaprimer method for site-directed mutagenesis is rapid, simple and highly efficient, and the success rate of mutagenesis could reach 100%. Furthermore, this method is suitable for routine application in molecular cloning.

Asian Continental Ancestry Group ; genetics ; DNA Primers ; Globins ; genetics ; Humans ; Mutagenesis, Site-Directed ; Polymerase Chain Reaction ; methods ; beta-Thalassemia ; ethnology ; genetics

OBJECTIVETo establish a one-step four multiplex reverse transcription polymerase chain reaction (RT-PCR) method based on Homo-Tag Assisted Non-Dimer System (HAND) system for simultaneous detection of 4 diarrhea viruses of rotavirus, astrovirus, norovirus and sapovirus.

METHODSPrimers were designed according to the conserved genome sequence of the 4 viruses and the homologous tail sequences were added to the 5' end. The multiplex RT-PCR system was constructed by optimizing the PCR parameters such as the concentration of universal tag primer and genome-specific Homo-Tailed primers. The specificity, stability and sensitivity of the method were evaluated systematically.

RESULTSThe 4 multiplex RT-PCR methods based on HAND system was established successfully. Specificity analysis showed no cross reaction between the 4 diarrhea viruses. The sensitivity analysis showed detection limits for rotavirus, astrovirus, norovirus and sapovirus of 48, 1.92, 9.6 and 48 pg per reaction, respectively.

CONCLUSIONThe established HAND system-based multiplex RT-PCR assay allows simple, rapid, specific, sensitive, and stable for detection of the 4 common diarrhea viruses at low costs and is suitable for application in general medical laboratories.

Astroviridae ; genetics ; isolation & purification ; Diarrhea ; virology ; Feces ; virology ; Humans ; Multiplex Polymerase Chain Reaction ; methods ; Norovirus ; genetics ; isolation & purification ; RNA, Viral ; isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Rotavirus ; genetics ; isolation & purification ; Sapovirus ; genetics ; isolation & purification

OBJECTIVETo investigate the gene frequencies and mutation patterns of alpha thalassemia (alpha-thal) and beta thalassemia (beta-thal) in Liuzhou city of Guangxi Zhuang Autonomous Region.

METHODSCluster sampling was used. A total of 1 028 of umbilical blood samples were collected for a prevalence study of alpha-thal and a total of 1 312 healthy young people when receiving pre-marriage consultation were recruited for a beta-thal prevalence survey. Individuals live in city or town area of Liuzhou. A complete blood count as well as hemoglobin electrophoresis analysis were done in all of samples for phenotyping of alpha and beta-thals. Those with Hb Bart's for alpha-thal indicator and those with both microcytosis (MCV < 85 fl) and elevated levels of Hb A(2) (>/=4.0%) for beta-thal were further studied by DNA analysis. PCR-based methodologies were used to characterize the mutation contributions of alpha and beta-thals. All the subjects were tested for the state of carrying beta-thala alleles for evaluating the situation of the compound heterozygotes of alpha-thal with beta-thal.

RESULTSOf 1 028 random samples of umbilical blood screened, 112 of subjects were defined to be the gene carriers of alpha-thal. The alpha-thal carrier rate was as high as 11.19% including 3 compound heterozygotes. Five well-known types of alpha-thal alleles were detected with gene contributions of 37.4% (--(SEA) deletion), 31.3% (-alpha(3.7) deletion), 17.4% (-alpha(4.2) deletion), 12.1% (alpha(CS)alpha mutation), and 0.9% (alpha(QS)alpha mutation), successively. Of the 1 312 adult specimens studied, 89 with beta-thal including 14 of the compound higher Hb F subjects were detected. All of the 89 phenotypic beta-thal carriers had the mutations in the beta-globin gene, making the overall prevalence 6.78%. The commonly seen three mutations, beta CD41 - 42 (-CTTT) frameshift, beta CD17 (T-A) nonsense mutation and beta-28 (A-G) promoter variation were accounted for 90% of the beta-thal alleles in Liuzhou. Of these beta-thal subjects, 16 (accounting for 18%) were found to be the compound heterozygosity for a beta-thal and an alpha-thal with 9 different types of gene defects with a detection rate 1.22%.

CONCLUSIONData from ecidation of alpha and beta-thal gene frequencies and mutation spectrum in Liuzhou city was useful for genetic counselling and prenatal diagnosis of this disease.

Adult ; China ; epidemiology ; Female ; Gene Frequency ; Genetic Counseling ; Humans ; Male ; Prevalence ; alpha-Thalassemia ; epidemiology ; genetics ; beta-Thalassemia ; epidemiology ; genetics

OBJECTIVETo develop a simple, rapid, accurate, and cost-effective single-0tube multiplex polymerase chain reaction (PCR) assay, which could be used for molecular screening and prenatal diagnosis, for detection of three commonest deletional alpha-thalassemias (-- (SEA), -alpha (3.7) and -alpha (4.2)) in Chinese population.

METHODSFour groups of primers were designed on the basis of gap-PCR, and the PCR reaction condition was optimized systematically with the purpose of amplifying effectively specific DNA fragments that are indicative of the respective genotypes of these three deletional alpha thalassemias. In addition, a pair of primers was designed to amplify LIS1 3' untranslated region (UTR) fragment for use as a separate control for amplification running. A total of 72 blood and prenatal archival DNA samples with various known alpha thalassemia genes or normal alpha globin gene sequence that had been confirmed by Southern blotting analysis or DNA sequencing were collected to test the specificity of this assay by blind analysis. In addition, DNA samples from nine couples at high risk of alpha thalassemia were also analyzed to evaluate the reliability of this technique in prenatal implementation.

RESULTSHomozygote, heterozygote and double heterozygote of the three commonest deletional alpha thalassemias were well detected simultaneously by this established method. For normal allele, a 2.4 kb amplified band as a systematic control and an alpha (2) gene-specific amplicon of 1.8 kb were produced. Besides the two amplified fragments of normal allele, it was found that a 1.3 kb, a 2.0 kb or a 1.6 kb amplified band could be simultaneously shown for representing --(SEA), -alpha (3.7) and -alpha (4.2) alleles, respectively, in the heterozygous states. In a blind test, this technique accurately detected 100% of the DNA samples previously characterized by Southern blotting or DNA sequencing, and it was successfully applied to prenatal diagnosis of alpha thalassemia in nine at-risk families.

CONCLUSIONThe single-tube multiplex PCR protocol presented in this study is easy-to-handle, rapid, reliable and is cost-effective for detecting --(SEA), -alpha (3.7) and -alpha (4.2) chromosomes, and it is suitable for large-scale population screening and for rapid molecular genotyping in clinics.

Asian Continental Ancestry Group ; genetics ; China ; Female ; Heterozygote ; Homozygote ; Humans ; Polymerase Chain Reaction ; methods ; Pregnancy ; Prenatal Diagnosis ; Reproducibility of Results ; Sensitivity and Specificity ; alpha-Thalassemia ; diagnosis ; ethnology ; genetics