A Simple Method for Detection of Multiple Chemical-Specific IgGs in Serum Based on Dot Blotting

Plastic resins are known to cause occupational allergies. Therefore, serum-specific antibodies against plastic resins have been widely investigated as diagnostic markers for occupational allergies. In this study, we aimed to establish a convenient method for detection of multiple chemical-specific IgG antibodies in human serum based on dot blot analysis. Toluene diisocyanate (TDI), phthalic anhydride (PA), and formaldehyde (FA), which are frequently used to synthesize various resins, reacted well with lysine residues of human serum albumin (HSA) under alkaline conditions. Native polyacrylamide gel electrophoresis (PAGE) showed that the structures of chemical adducts of HSA were different from those of native HSA. Therefore, we performed dot blot assays using these adducts as artificial antigens. Serum samples from workers at plants utilizing plastic resins strongly reacted with TDI, PA, and FA adducts in HSA, while reduced signals were detecting using the serum from unexposed workers. These results suggested that dot blot assays using chemical-HSA adducts as antigens could be beneficial for simultaneously measuring multiple chemical-specific IgGs.

番茄斑萎病毒外壳蛋白原核表达及Dot-blotELISA检测方法的建立

将番茄斑萎病毒(Tomato spotted wilt virus,TSWV)全长外壳蛋白基因亚克隆到原核表达载体pET-32a中,经限制性酶切分析及测序结果表明插入方向正确且阅读框架无移码突变.将重组表达质粒转化大肠杆菌BL21(DE3),SDS-PAGE分析结果证实IPTG可诱导1个分子量约为48 kDa的融合蛋白表达.利用6×His标签单抗和TSWV FoPaTs1多抗证实所表达的蛋白为TSWV外壳蛋白.以纯化的重组外壳蛋白免疫小鼠,制备杂交瘤细胞培养上清单抗,并用杂交瘤细胞培养液上清建立了可靠、有效检测TSWV的Dot-blot ELISA方法.

Comparison of the Tellgenplex HPV DNA test with the PCR-reverse dot blot assay for human papillomavirus genotyping

Objective: To access the performance of the Tellgenplex human papillomavirus(HPV) DNA test compared to the polymerase chain reaction-reverse dot blot(PCR-RDB) assay for the HPV genotyping.Methods: Sixty cervical swab samples were genotyped by the Tellgenplex HPV DNA test and the PCR-RDB assay.The Tellgenplex HPV DNA test and the PCR-RDB assay can detect 26 and 23 HPV genotypes, respectively.Each sample showed discrepancy was genotyped using sequencing.Results: The percent agreement between the two tests ranged from 83.3% to 100.0% according to different genotype.This showed perfect agreement(>0.81) for high-risk HPV genotypes(35, 39, 45, 53, 56, 59, 66, 68, and 82), substantial agreement(>0.65) for high-risk HPV genotypes(16, 18, 33, 52, and 58) and low-risk HPV genotype 43 between the two assays by the kappa analysis.The positive rates of the two assays for frequent HPV genotypes(16, 35, 39, 45, 52, 53, 58, 59, 66, and 82) were not statistically different, but the PCR-RDB assay showed higher positive rates than the Tellgenplex HPV DNA test for HPV genotypes 81(P<0.05).As for more than 10 positive results by the Tellgenplex HPV DNA test and/or the PCR-RDB assay, the PCR-RDB assay showed higher relative sensitivity and specificity than the Tellgenplex HPV DNA test for the three HPV genotypes(16, 52, and 81).All HPV genotypes that can be detected by only the Tellgenplex HPV DNA test(HPV genotypes 44 and 55) were confirmed by sequencing.Conclusions: In conclusion, our results demonstrated that the PCR-RDB assay which can detect more multiple HPV genotypes in each specimen shows higher relative sensitivity and specificity than the Tellgenplex HPV DNA test, which makes it a better option for routine clinical use.

Rapid Detection of Rifampin-resistant Clinical Isolates of Mycobacterium tuberculosis by Reverse Dot Blot Hybridization

Objective A PCR-reverse dot blot hybridization （RDBH） assay was developed for rapid detection of rpoB gene mutations in ＇hot mutation region＇ of Mycobacterium tuberculosis （M. tuberculosis）. Methods 12 oligonucleotide probes based on the wild-type and mutant genotype rpoB sequences of M. tuberculosis were designed to screen the most frequent wild-type and mutant genotypes for diagnosing RIF resistance. 300 M. tuberculosis clinical isolates were detected by RDBH, conventional drug-susceptibility testing （DST） and DNA sequencing to evaluate the RDBH assay. Results The sensitivity and specificity of the RDBH assay were 91.2% （165/181） and 98.3% （117/119）, respectively, as compared to DST. When compared with DNA sequencing, the accuracy, positive predictive value （PPV） and negative predictive value （NPV） of the RDBH assay were 97.7% （293/300）, 98.2% （164/167）, and 97.0% （129/133）, respectively. Furthermore, the results indicated that the most common mutations were in codons 531 （48.6%）, 526 （25.4%）, 516 （8.8%）, and 511 （6.6%）, and the combinative mutation rate was 15 （8.3%）. One and two strains of insertion and deletion were found among all strains, respectively. Conclusion Our findings demonstrate that the RDBH assay is a rapid, simple and sensitive method for diagnosing RIF-resistant tuberculosis.

用抑制消减杂交法分离巴西橡胶树胶乳特异表达基因

采用巴西橡胶树常割树胶乳的Poly(A+)RNA为Tester,叶片Poly(A+)RNA为Driver,通过抑制消减杂交法(suppressivesubtractivehybridization,SSH)构建了一个胶乳特异表达基因差减文库,通过菌落PCR及反式NorthernDot-Blot筛选鉴定阳性差异片段,获得79个胶乳特异表达阳性克隆,部分阳性克隆还通过NorthernBlot杂交及RT-PCR进一步验证。随机挑选部分阳性克隆序列比较分析,结果表明有部分克隆与巴西橡胶树中已知的基因相同,而多数克隆在巴西橡胶树中是新发现,它们与橡胶生物合成、物质代谢运输、信号传导和形态建成等相关。

柚(Citrus grandis Osbeck)单染色体LA-PCR-AFLP分子标记体系的建立

通过微分离和微克隆技术,对柚单染色体LA-PCR-AFLP技术体系中反应程序、模板浓度等关键参数进行了优化和引物筛选。结果表明:退火温度较高的程序TD3[退火梯度:72℃→65℃30 s(-0.5℃/cyc)]扩增效果较好;稀释100倍(10 ng/μL)为最适模板量;10个引物对组合扩增多态性较好。对22条单染色体的AFLP多态性分析和dot-blot杂交验证,表明较好地构建了柚单染色体LA-PCR-AFLP体系。这为柚单染色体的识别和文库构建提供了试验基础。

Profile, spectrum and significance of hepatitisB virus genotypes in chronic HBV-infected patients in Yunnan, China

BACKGROUND: There are significant variations in the geographical distribution of hepatitis B virus (HBV) genotypes throughout the world, and some genotypes are associated with different clinical outcomes. Eight genotypes of human HBV (designated A-H) have been reported. The present study was designed to examine the distribution of HBV genotypes among patients at various stages of chronic type B liver disease in Yunnan Province, China, and to explore its significance and the relationship of HBV genotype with gender and age, clinical spectrum of chronic HBV infection, and viral replicative activity. METHODS: Serum samples from 126 patients with chronic HBV infection from Yunnan Province, including 26 chronic asymptomatic HBV carriers (ASC), 61 patients with chronic hepatitis B (CHB) (21 mild, 30 moderate and 10 severe), 20 patients with chronic fulminant hepatic failure (CFHF), 12 patients with HBV-related liver cirrhosis (LC) and 7 patients with HBV-related hepatocellular carcinoma (HCC) were analyzed using reverse dot blot (RDB) methodology, which is based on the reverse hybridization principle for HBV genotyping. The relations of HBV genotype with gender and age, clinical patterns, and serological data of the patients were analyzed. RESULTS: In this series, genotypes A, B, C, and D were found. 38.1% patients (48/126) belonged to B, 54.8% (69/126) to C, 0.8% (1/126) to D, 1.6% (2/126) to a mixture of B and C, and 1.6% (2/126) to a mixture of A and C. 3.2% patients (4/126) had unknown genotypes. No other genotypes (E, F, G, and H) were found. Genotypes B and C were predominant. There was a statistically significant difference in the distributions of genotypes C and B (chi(2)=7.04, P=0.008), and C was the dominant genotype in all patient categories. The rate of genotype B in the mild CHB group was significantly higher than that in the moderate and severe groups (chi(2)=12.16, P=0.0001; chi(2)=11.98, P=0.001, respectively), the ASC group (chi(2)=5.46, P=0.02), the CFHF group (chi(2)=5.53, P=0.019), and the LC/HCC group (chi(2)=12.13, P=0.001). The rate of genotype C in the LC/HCC group and the severe CHB group were significantly higher than that in the mild group (chi(2)=9.95, P=0.002; chi(2)=8.78, P=0.003, respectively). HBV DNA positivity and HBeAg positivity were higher in genotype C than in genotype B (chi(2)=9.81, P=0.002; chi(2)=3.85, P=0.05, respectively). The prevalence of genotype C showed an increasing trend in lowest-, middle- and highest-level groups of HBV replication (25.0%, 70.0%, and 55.6%, respectively); in contrast, the prevalence of genotype B showed an opposite trend in the same order (62.5%, 30.0%, and 37.0%, respectively). The rate of genotype C in the highest-level group of HBV replication was higher than genotype B (chi(2)=7.45, P=0.006). The rate of genotype C in the over-30 age group was higher than that in the below-30 age group (chi(2)=3.7, P=0.05). There was no difference between the sexes (P>0.05). More severe liver damage was found in genotype C than in genotype B (P<0.05). CONCLUSIONS: The predominant HBV genotypes in chronic HBV-infected patients are B and C, and C is the most prevalent genotype in Yunnan Province, China. HBV genotype C is associated with the development of more severe liver disease and a higher level of HBV viral replication, and genotype B has a relatively good progress.

Differential gene expression between asymptomatic HBV carriers and normal adults

BACKGROUND: There are more than 100 million wa symptomatic HBV carriers (ASCs) in China and they are at a high risk of developing liver disease, which creates a serious health problem. But more than 90% of normal adults clear virus from primary HBV infection, so the difference of gene expression between ASCs and normal adults determines the differential outcomes. To identify differentially expressed genes in ASCs compared to normal adults, we used suppression subtractive hybridization to compare gene expression. METHODS: cDNA subtracted libraries were constructed by suppression subtracted hybridization from peripheral blood monocytes of ASCs and normal adults, the subtracted clones were prescreened by dot blot hybridization, and the levels of genes of interest were confirmed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: One hundred and two and 96 positive clones were acquired from the A-N and N-A libraries, respectively, and 14 and 11 clones were identified by dot blot hybridization in the A-N and N-A libraries. Two genes were confirmed as differentially expressed between a set of ASC and normal adult samples by real-time RT-PCR. CONCLUSIONS: Differentially expressed genes in peripheral blood mononuclear cells between ASCs and normal adults were isolated by suppression subtractive hybridization, and included some new genes. Of the upregulated genes in ASCs, checkpoint suppressor I is associated with DNA damage-induced cell cycle arrest. Perforin I is associated with inflammation. The information about such alterations in gene expression could be useful for elucidating the mechanisms of ASC pathogenesis.

Effect of Jianpiyiqi Prescription on the Expression of Heat Shock Proteins in Acetic Acid-induced Chronic Gastric Ulcer Rats

To investigate the effect of Jianpiyiqi prescription on the expression of heat shock proteins and the changes of HSPs in gastric ulcerated rats, the rat model of chronic gastric ulcer was induced by acetic acid. The SABC immunohistochemical method was used to observe HSP70 of mucosa around the gastric ulcer. Imaging analysis was performed. Western dot blot was used to detect HSPs contents in the plasma and gastric mucosal homogenate in each group. The results showed that HSP70 contents of the mucosa around the gastric ulcer in the model group and ranitidine treated group were increased as compared with control group . Jianpiyiqi could increase the expression of HSP70 of the mucosa around the gastric ulcer further as compared with that in the model group and ranitidine treated group . The HSP70 contents in the serum and mucosa in the model group and ranitidine treated group were increased as compared with control group ( P <0.01, P <0 05 respectively). HSP70 of serum and mucosa in the Jianpiyiqi treated group was higher than in the model group and ranitidine treated group ( P <0.05, P <0.01 respectively).It was concluded that HSP might play a role in the process of pathophysiology of gastric ulcer. Jianpiyiqi could enhance gastric ulcer healing through the protective mechanism of HSPs.

Isolation, Cloning, and Identification of Expressed Sequence Tags from the Backfat Tissue in Duroc and Tongcheng Pigs by mRNA Differential Display

mRNA differential display technique was performed to discuss the differential expression of genes in fat tissue between introduced European and Chinese indigenous pigs. Four anchor primers in combination with five arbitrary primers （20 sets in total） were used and nearly 300 bands were observed in polyacrylamide gel, among which 29 differential display bands were obtained. Twelve of 29 cDNA fragments were identified using reverse Northern dot blot, and subsequently cloned and sequenced. Eight of 12 cDNAs had no matches in GenBank and were submitted to GenBank, and the other 4 showed similarity to identified genes from GenBank. Three among 8 novel ESTs were selected to be further identified by semiquantitative RT-PCR. In our experiment, silver staining DDRT-PCR and DIG primer DNA labeling reverse Northern dot blot were used to avoid radioactive pollution. The result showed that the expressions of 5 among 8 novel ESTs were stronger in the backfat of Tongcheng pigs and the others were weaker than that in Duroc pigs. These novel ESTs were prepared for selecting genes related to adipose cells.

Carrier Screening and Prenatal Gene Diagnosis of β-thalassemia by PCR-RDB Technique

In order to identify the distribution of gene types of β-thalassemia and reduce the birthrates of β-thalassemia major in Guiyang area, 1054 pregnant women and their spouses from Affiliated Hospital, Guiyang Medical College were screened. The positive samples were analyzed with polymerase chain reaction and reverse dot blot method (PCR-RDB). When both partners were heterozygous identified as carriers for β- thalassemia, the risk of having a fetus who was homozygous or compound heterozygous was 2.66 %; the ratio of male to female was 1/1.15. Seven types of mutation were identified. CD17 and CD41-42 were dominant among them. Among the 4 cases subject to prenatal gene diagnosis, one fetus was completely normal and 3 fetuses were diagnosed as having β-thalassemia major (1 homozygous and 2 compound heterozygous). The fetuses diagnosed as β-thalassemia major were selectively terminated within two weeks. It was concluded that the birthrate of β-thalassemia major in Guiyang area was reduced and the target of improving birth outcome and child development has been achieved.

Expression and Cellular Localization of Interleukin-6 mRNA in Ovariectomized Rats

In order to observe the expression and cellular localization of interleukin-6 (IL-6) mRNA in bone tissue, ovaria of the rats were excised to develop osteoporosis model. The expression of IL-6 mRNA in bone tissues was detected by using dot blot hybridization assay and the cells producing IL6 identified and localized by using in situ hybridization respectively. The results showed that the expression of IL-6 mRNA was significantly increased in the ovariectomized rats as compared with that in normal control rats and strong IL-6 mRNA hybridization signals were detected in lining cells, osteoblasts and osteocytes. It was suggested that loss of ovarian function induced in vivo osteoblast lineage increased IL-6 mRNA expression. IL-6 might play important roles in the development of bone loss following ovariectomy.

Temporal analysis of mRNA expression of endogenous TGFβ And its typeⅠ,typeⅡreceptor on burn wounds

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Screening of Species-specific DNA Probes for Identification of Fallopia multiflora

To screen species-specific DNA probes for identification of Fallopia muhiflora, the genomic DNA （gDNA） suppression subtraction hybridization （SSH） between F. muhiflora and F. muhiflora var. ciliinervis was firstly performed. The obtained differential gDNA fragments by SSH were then hybridized with gDNA ar- rays consisting of multiple whole genomes of several species （adulterants and/or closely related species of F. muhiflora） and four differential fragments were screened uniquely representing F. muhiflora, which could be used as F. muhiflora species-specific probes. The screened DNA probes were tested by reverse dot blot hybridization and the results demonstrated that these probes could be used reliably to identify F, muhiflora. The species-specific DNA probes obtained in this study exhibited broad application prospects in the preparation of gene chips for identifying Chinese traditional medicines and the authentication of germplasm re- sources and crude drugs of F. muhiflora.

Detection of Human Papillomavirus Type 16 DNA in Cervical Carcinomas

Accurate typing of the different human papillomavirus types is csscntial in view of the differ-ent pathological potential of the common virus types of human papillomavirus （HPV） present in thecervix. We have developed hybridization, washing and autoradiography conditions that minimize thecross-hybridization among different specific types of HPV so as to allow clear - cut type assignmentthrough practical dot blot hybridization technique using nylon membrane and 35S - labeled HPV - 16DNA probe. Under these conditions seventeen of thirty （56.7%） of squamous cell carcinomas of thecervix uteri obtained from Tianjin women were detected in the presence of HPV - 16 DNA.

Construction of white spot syndrome virus (WSSV) whole genome phage display library

A rebuilt vector pCANTAB 5 EE was obtained by inserting a 34 bp double-stranded oligonucleotide which contained a EcoRV recognition sequence into pCANTAB 5 E. White spot syndrome virus （WSSV） genome DNA was fragmented by sonication to isolate fragments mainly in the range of 0.8 ～2.0 kb, then the fragments were blunt-ended with T4 DNA polymerase and cloned into the EcoRV site of pCANTAB 5 EE. The primary recombinant clone of the library was 3.0 × 10^5.Colony PCR of random selected recombinants showed that the size of the inserts was 0.12 ～ 1.77 kb. After the whole library recombinant phages infected Escherichia coli HB2151 cells, the extracellular and periplasmic extracts were dropped on PVDF membranes to perform dot blot, using polyclonal mouse anti-VP24 serum,anti-WSV026 serum,anti-WSV063 serum,anti-WSV069 serum,anti-WSV112 serum, anti WSV238 serum,anti-WSV303 serum and anti-VP26 serum as the primary antibody, respectively. The results showed that the display library could express the viral proteins.

Accuracy of a reverse dot blot hybridization assay for simultaneous detection of the resistance of four anti-tuberculosis drugs in Mycobacterium tuberculosis isolated from China

Background:Drug resistant tuberculosis poses a great challenge for tuberculosis control worldwide.Timely determination of drug resistance and effective individual treatment are essential for blocking the transmission of drug resistant Mycobacterium tuberculosis.We aimed to establish and evaluate the accuracy of a reverse dot blot hybridization(RDBH)assay to simultaneously detect the resistance of four anti-tuberculosis drugs in M tuberculosis isolated in China.Methods:In this study,we applied a RDBH assay to simultaneously detect the resistance of rifampicin(RIF),isoniazid(INH),streptomycin(SM)and ethambutol(EMB)in 320 clinical M.tuberculosis isolates and compared the results to that from phenotypic drug susceptibility testing(DST) and sequencing.The RDBH assay was designed to test up to 42 samples at a time.Pearson's chi-square test was used to compute the statistical measures of the RDBH assay using the phenotypic DST or sequencing as the gold standard method,and Kappa identity test was used to determine the consistency between the RDBH assay and the phenotypic DST or sequencing.Results:The results showed that the concordances between phenotypic DST and RDBH assay were 95%for RIF,92.8%for INH,84.7%for SM,77.2%for EMB and the concordances between sequencing and RDBH assay were 97.8%for RIF,98.8%for INH,99.1%for SM,93.4%for EMB.Compared to the phenotypic DST results,the sensitivity and specificity of the RDBH assay for resistance detection were 92.4 and 98.5%for RIF,90.3 and 97.3%for INH,77.4 and 91.5%for SM,61.4 and 85.7%for EMB,respeaively;compared to sequencing,the sensitivity and specificity of the RDBH assay were 97.7 and 97.9%for RIF,97.9 and 100.0% for INH,97.8 and 1OO.O% for SM,82.6 and 99.1%for EMB,respectively.The turnaround time of the RDBH assay was 7 h for testing 42 samples.Conclusions:Our data suggested that the RDBH assay could serve as a rapid and efficient method for testing the resistance of M. tuberculosis against RIF,INH,SM and EMB,enabling early administration of appropriate treatment regimens to the affected drug resistant tuberculosis patients.

Effect of 8-bromo cyclic AMP on neuron specific enolase, heat shock protein, nitric oxide, nitric oxide synthase and nitric oxide synthase mRNA in human retinoblastoma HXO Rb44 cells and cell differentiation

Objective To study the effect of 8 bromo cyclic AMP (8 Br cAMP) on nitric oxide synthase (NOS) mRNA, NOS and nitric oxide (NO) product, heat shock protein (hsp)70 and neuron specific enolase (NSE) in human retinoblastoma HXO Rb44 cells and the effect related to cell differentiation Methods Cultured human retinoblastoma HXO Rb44 cells were divided into two aliquots One was cultured with 2×10 5 ?mol/L of 8 Br cAMP for 24 hours as the experiment group; the other was treated with no 8 Br cAMP as the control group The cell suspensions in concentration of 1×10 7/ml in both groups were dropped onto the nitrocellulose membrane (NCM) The NOS mRNA was detected with the biotin labeled NOS cDNA probe by RNA dot blot The NOS activity was detected by protein dot blot The immunoreactivity (IR) of hsp70 and NSE was detected by protein dot blot The NO was detected by nitrate reductase method NCM specimens were analyzed by a TLC scanner for detection of the dot blot signal intensity Results The signals of NOS mRNA, NOS activity, hsp70 IR, NSE IR, and NO content in the experiment group were higher than those in the control group ( P <0 05-0 01) Conclusions 8 Br cAMP could increase NO product and the expression of NOS mRNA, NOS , NSE and hsp70 The results indicate that 8 Br cAMP could facilitate synthesis of NO in the neuroblastoma HXO Rb44 cells which could have tendency toward neuron development, suggesting that the increased hsp70, NO and NOS may involve cell differentiation of the retinoblastoma HXO Rb44

Development of a Low-cost Polymerase Chain Reaction-based Method for Studying Differentially Expressed Genes in Developing Rice Leaves

Gene expression studies are important for revealing gene functions putatively involved in biological processes. We were interested in identifying differentially expressed genes during leaf development in rice. We combined the RNA arbitrarily primed-polymerase chain reaction （RAP.PCR） and dot blot hybridization methods to screen a rice leaf primordium cDNA library. Three developmental stages during vegetative growth were examined. The cDNA clones showing different hybridization patterns were further analyzed and verified. Here we demonstrate that the combination of RAP-PCR and dot blot hybridization could provide an efficient and relatively low-cost cDNA library screening approach to discover genes not previously known to be associated with leaf development in rice, We believe that the findings described here will help to elucidate the molecular mechanism（s） underlying the developmental processes of rice leaf