One-step Reverse Transcriptase Polymerase Chain Research Articles

Molecular and Serological Investigation of Infectious Bronchitis Virus in the East Shewa, Central Ethiopia.

Infectious bronchitis (IB) is an economically important disease in poultry with worldwide distribution. The occurrence of IB has been reported both in commercial and backyard poultry in Ethiopia, although comprehensive information lacks available prevalence of the disease and the circulating serotypes. A cross-sectional study was conducted from November 2021 to June 2022 in seven commercial farms found in East Shewa, Central Ethiopia. Serological assay using indirect ELISA, virus isolation techniques in embryonated eggs, and molecular techniques such as one-step reverse transcriptase polymerase chain reaction (RT-PCR) and nested polymerase chain reaction (PCR) targeting a 466 bp S1 gene were employed. A total of 196 blood samples, 7 pools (35) of swab samples, and 5 pools of tracheal samples were investigated. The results of serological analysis revealed that 97.96% (192/196; 95% CI: 94.86-99.44) of the sera samples were found to be positive for antibodies against IBV. Out of the 7 pools of swab and 5 pools of tracheal tissue samples analyzed using RT-PCR 33.3% (4/12) of them gave positive results all from swab samples. The RT-PCR-positive samples were subjected to a nested PCR yielding 295bp and 154bp indicating the circulation of Mass and 793/B (4/91) strains of IBV, respectively. The 12 pools of samples inoculated into embryonated egg showed cytopathic changes such as congestion, bleeding, and deformation only after three passages. Two serotypes of IBV are circulating in Ethiopian chickens, and molecular identification of the Massachusetts serotype is the first report in Ethiopia. Further epidemiological investigation is needed in order to devise effective control measures.

Improvement of the qmosRT-PCR Assay and Its Application for the Detection and Quantitation of the Three Serotypes of the Novel Oral Polio Vaccine in Stool Samples.

Recently, genetically stable novel OPVs (nOPV) were developed by modifying the genomes of Sabin viruses of conventional OPVs to reduce the risk of reversion to neurovirulence and therefore the risk of generating circulating vaccine-derived polioviruses. There is a need for specific and sensitive methods for the identification and quantification of nOPV viruses individually and in mixtures for clinical trials and potentially for manufacturing quality control and environmental surveillance. In this communication, we evaluated and improved the quantitative multiplex one-step reverse transcriptase polymerase chain reaction (qmosRT-PCR) assay for the identification and quantification of nOPV viruses in samples with different formulations and virus concentrations and in virus-spiked stool samples. The assay was able to specifically identify at least 1 log10 CCID50/mL of each serotype in the presence of the two other serotypes at high concentrations (6-7 log10 CCID50/mL) in the same sample. In addition, the lowest viral concentration that the assay was able to detect in stool samples was 17 CCID50/mL for nOPV1 and nOPV2 viruses and 6 CCID50/mL for nOPV3. We also found high correlation between the expected and observed (by qmosRT-PCR) concentrations of spiked viruses in stool samples for all three nOPV viruses, with R-squared values above 0.95. The analysis of samples collected from an nOPV2 clinical trial showed that 100% of poliovirus type 2 was detected and few samples showed the presence of type 1 and 3 residuals from previous vaccinations with bOPV (at least 4 weeks prior vaccination with nOPV2), confirming the high sensitivity of the method. The qmosRT-PCR was specific and sensitive for the simultaneous identification and quantification of all three nOPV viruses. It can be used as an identity test during the nOPV manufacturing process and in evaluation of virus excretion in nOPV clinical trials.

Full-length coding sequence analysis of genome segments A and B of infectious bursal disease virus in Thailand: identification of Chinese-like and recombinant virus in the field

ABSTRACT Infectious bursal disease virus (IBDV) causes immunosuppression in chickens, with distinctive bursal lesions. Genomic sequence information is essential to understand the epidemiology of antigenically and/or pathogenically variable IBDV strains. The present study investigated the full-length coding sequences of genome segments A and B of IBDV circulating in Thailand during 2010–2015. Five clinical samples of pooled bursas obtained from five commercial chicken farms experiencing problems with IBD in five different provinces of Thailand were identified by using a one-step reverse transcriptase polymerase chain reaction. The full-length coding sequences of genome segments A and B of three and five isolates, respectively, were successfully characterized. Phylogenetic analysis showed that two groups of IBDV were circulating in Thailand. The first group was similar to strain HLJ-504, genotype A3B3, isolated in China, but it had some minor variations in both genome segments A and B. The second group, which had a recombination event in segment A, was clustered with genotype A1B1. Its segment A originated from the intermediate vaccine W2512-like strain and isolate 9109, whereas segment B shared characteristics of a classical strain without recombination events. This study characterized the full-length coding sequences of genome segments A and B of Thai IBDV and demonstrated the presence of recombinant IBDV in Thailand for the first time. Therefore, the molecular characterization of the full-length coding sequences of both genome segments of IBDV is essential to monitor the emergence of new variant viruses, which is important for effective control and appropriate vaccine development. RESEARCH HIGHLIGHTS For the first time, this work demonstrated a recombinant IBDV strain in Thailand. Two genogroups of IBDV were found in Thailand: including HLJ-504-like and recombinant virus. Analysis of the full coding sequence is essential for monitoring emerging variant IBDV.

Serological and Molecular Detection of Avian Influenza Virus ‎Hemagglutinin (H5) in Wild Birds in and around Zaria, Nigeria

Avian influenza (AI) has a worldwide distribution and affects domestic and wild birds, thus causing great economic losses to the poultry industry. This study was carried out to detect avian influenza virus H5 antibodies and nucleic acidin some wild birds [Laughing doves (Spilolepia senegalensis), Speckled pigeons (Columba guinea), Cattle egrets (Bubulcus ibis), Senegalese parrots (Poicephalussenegalus), Mallards (Anas platyrhynchos) and Geese (Anseranserini)] in Zaria and its environs, Kaduna State Nigeria. Sera were tested for avian influenza virus (AIV) H5 antibody using competitive enzyme linked immunosorbent assay (cELISA). Pooled oropharyngeal and cloacal swabs of each bird species (8-10 samples) were tested for AIV nucleic acidusing one-stepreverse transcriptase polymerase chain reaction (RT-PCR). Results revealed overall prevalence of 6.62% and 3.85% for AIV antibody and nucleic acid respectively. Based on species, AIV antibody was detected in laughing dove (10%), speckled pigeon (13.64%) and mallard (19.05%).Also, AIV antigen was detected in Senegalese parrot (20%).In conclusion, AIV antibody and antigen were detected in wild birds in Zaria, Nigeria. Thus, these species of birds could play significant roles in the spread of this virus to chickens. Therefore, measures to limit the interactions of these wild birds with chickens should be implemented to minimize the spread of AI.

Comparative Neuropathology of Major Indian Bluetongue Virus Serotypes in a Neonatal BALB/c Mouse Model

Bluetongue virus (BTV) is neurotropic in nature, especially in ruminant fetuses and in-utero infection results in abortion and congenital brain malformations. The aim of the present study was to compare the neuropathogenicity of major Indian BTV serotypes 1, 2, 10, 16 and 23 by gross and histopathological lesionsand virus distribution in experimentally infected neonatal BALB/c mice. Each BTV serotype (20μl of inoculum containing 1×105 tissue culture infectious dose [TCID]50/ml of virus) was inoculated intracerebrally into 3-day-old mice, while a control group was inoculated with mock-infected cell culture medium. Infection with BTV serotypes 1, 2 and 23 led to 65-70% mortality at 7-9 days post infection (dpi) and caused severe necrotizing encephalitis with neurodegenerative changes in neurons, swelling and proliferation of vascular endothelial cells in the cerebral cortex, cerebellum, midbrain and brainstem. In contrast, infection with BTV serotypes 10 and 16 led to 25-30% mortality at 9-11 dpi and caused mild neuropathological lesions. BTV antigen was detected by immunohistochemistry, direct fluorescence antibody technique and confocal microscopy in the cytoplasm of neuronal cells of the hippocampus, grey matter of the cerebral cortex and vascular endothelial cells in the midbrainand brainstem of BTV-1, -2, -10, -16 and -23 infected groups from 3 to 20 dpi. BTV nucleic acid was detected in the infected brain tissues from as early as 24h up to 20 dpi by VP7 gene segment-based one-step reverse transcriptase polymerase chain reaction. This study of the relative neurovirulence of BTV serotypes is likely to help design suitable vaccination and control strategies for the disease.

Virological Perspectives and Characterization of Seasonal Influenza B Viruses in Mumbai

Acute respiratory infections caused by influenza viruses are major life-threatening infections affecting the global population. Information on influenza viruses in Mumbai region in India is limited. With an estimated population above one crore, it becomes imperative to identify the predominant circulating strains, seasonal evolution, epidemiology and impact of influenza virus infection on the public health of Mumbai. The present study is aimed to isolate and identify the prevalent influenza B virus strains circulating in the Mumbai region. Virological analysis of 482 throat and nasal samples from patients presenting with suspected influenza-like illness was conducted from January to December 2012 at a research institute in Mumbai. Isolation of positive influenza B samples was performed on Madin Darby Canine Kidney (MDCK) cell line. The isolates were further characterized by hemagglutination inhibition assay and one-step reverse transcriptase polymerase chain reaction. Among all the specimens tested, 71% were positive for influenza virus. The positivity for influenza B virus, influenza A (H1N1) pdm 09 and seasonal influenza A virus was 38%, 22% and 11% respectively. Of the 80 influenza B specimens cultured on MDCK, virus was isolated from 38 (48%) samples. Influenza B/Wisconsin/1/2010-like serotype belonging to the B/Yamagata/18/88 lineage was the predominant serotype identified. Influenza was a significant public health concern in the Mumbai region during the study period. Young children were the most affected age group. The study data generated will help understand the epidemiology of the disease in the Mumbai region as well as generate information for global influenza surveillance.

Neutrophil gelatinase-associated lipocalin (NGAL) as a potential biomarker for early detection of acute renal failure

Neutrophil gelatinase-associated lipocalin (NGAL) is a biomarker that can potentially be used for the detection of acute renal failure earlier and more accurately than current standard examinations, such as serum levels of creatinine and urea which are less sensitive and less specific. The objective of this research was to assess the ability of NGAL as a biomarker of acute renal failure by comparing the levels of serum urea and creatinine in rats with induced acute renal failure. Twelve male Wistar rats (Rattus norvegicus), 2 months of age with a body weight (BW) of 150–200 g, were allocated into two groups. Group I (n = 2), as the control group, were injected with sterile distilled water 10 mL/kg BW intramuscularly and group II (n = 10) were injected with 50 % glycerol 10 mL/kg BW intramuscular, the rats in all group had been fasting 12 h before injection. In group II, two rats were selected at each time point (0, 6, 12, 24 and 48 h after injection) bloods collected for analysis of urea and creatinine concentration and identification of NGAL and they were necropsied and the kidney removed for histopathological analysis and NGAL identification. The levels of creatinine and urea were analysed by independent t test. Identification of NGAL was conducted by one-step reverse transcriptase polymerase chain reaction and sequencing. Results showed that the glycerol injection significantly elevated the levels of urea at 6, 12 and 24 h compared to the control group (p < 0.05). Creatinine levels were significantly increased at 24 h compared to the control group (p < 0.05). The expression of NGAL in kidney and blood samples could be detected at 6, 12 and 24 h after induction. In conclusion, the blood NGAL could be detected before increasing of creatinine levels and could be used as an early detection of acute renal failure.

Association of oral tumor suppressor gene deleted in oral cancer-1 (DOC-1) in progression of oral precancer to cancer

IntroductionDeleted in oral cancer-1 (DOC-1), a highly conserved tumor suppressor gene, encodes a specific cyclin-dependent kinase 2-associated protein 1 (p12DOC-1). The protein DOC-1 arrests the cells in the G1 phase of the cell cycle and regulates DNA replication in the S phase of the cell cycle. It is known that DOC-1 is downregulated in head and neck cancer. Hence, the aim of the study was to analyze the messenger RNA (mRNA) expressions of DOC-1 in patients with oral lichen planus (OLP), leukoplakia (LPK), oral submucous fibrosis (OSF), and oral squamous cell carcinoma (OSCC) in comparison to normal control subjects, and these were correlated with differential oral habits and duration of exposure. Materials and methodsSemiquantitative one-step reverse transcriptase polymerase chain reaction (RT-PCR) was used to assess the potential role of DOC-1 in differential oral conditions. ResultsThe expression of DOC-1 at the transcriptional level was found to be consistently reduced (39.13%) or non-detectable (60.87%) in OSCC cases; LPK cases also showed reduced (26.19%) or non-detectable (59.52%) DOC-1 expression, and overexpression of DOC-1 was observed in patients with OSF (42.3%) and OLP (35.29%) as compared to the normal control groups. A marked reduction in DOC-1 expression was observed in patients who smoke bidi and chew tobacco compared to patients who smoke cigarettes and chew pan. An overall reduced expression of DOC-1 with an increase in the duration of exposure was observed. ConclusionsThe overexpression of DOC-1 in oral premalignant disease groups with reduced or loss of expression in OSCC groups suggests its association in the progression of oral carcinogenesis, suggesting DOC-1 to be an important prognostic indicator for oral cancer.

Detection of BCR–ABL using one step reverse transcriptase- polymerase chain reaction and microchip electrophoresis

One-step reverse transcriptase polymerase chain reaction (RT-PCR) coupled with microchip electrophoresis (MCE) was established to analyze BCR–ABL fusion gene. The use of one-step RT-PCR could simplify the RT-PCR procedure and thus reduced the risk of contamination and sample consumption. This method also enhanced the sensitivity for amplified target DNA and dramatically shorted the analysis time. Moreover, this assay can simultaneously identify b2a2 and b3a2. Orthogonal array design, which can investigated mutual effects of PCR parameters, was used to optimize the reaction system. This approach was highly effective, reproducible and sensitive, and would be suitable for the determination of BCR–ABL in clinic diagnosis.

Effect of recombinant adeno-associated BMP-4/7 fusion gene on the biology of BMSCs

The aim of the study was to investigate the effect of recombinant adeno-associated virus (AAV) with morphogenetic protein 4/7 fusion gene on the ossification of rabbit bone marrow stromal cells (BMSCs). The genes BMP-4 and BMP-7 were obtained through one-step reverse transcriptase polymerase chain reaction from human placental cells. The BMP-4/7 fusion gene was generated through recombination. The rabbit BMSCs were transfected with the recombinant AAV vectors carrying AAV-BMP-4/7 with various multiplicity of infection (MOI) values. Expression of fusion gene BMP-4/7 protein was determined by the ELISA method. The ossification of cells was evaluated by observing alkaline phosphatase (ALP) and osteocalcin (OC) activity after transfection for 7 and 14 days. We successfully constructed the recombinant AAV with the BMP-4/7 fusion gene. A 29-20 kDa protein was shown by SDS-PAGE electrophoresis following transfection with AAV-BMP-4/7. The fusion protein BMP-4/7 in BMSCs transferred by AAV showed a positive correlation with various MOI values. There was significantly higher ALP and OC activity in the AAV-BMP-4/7 transfection groups than in the AAV-EGFP groups (t(ALP)=896.88, P<0.001, t(OC)=543.24, P<0.01). The fusion gene BMP-4/7 is capable of enhancing the expression of BMPs and possesses significant ossification activity through AAV.

Recombinant adeno-associated virus BMP-4/7 fusion gene confers ossification activity in rabbit bone marrow stromal cells

The biological effects of transfection of an adeno-associated virus (AAV) vector with bone morphogenetic proteins 4 and 7 (BMP-4/7) fusion gene (AAV-BMP-4/7) were determined in rabbit bone marrow stromal cells (BMSCs). BMP-4 and BMP-7 genes were obtained through one-step reverse transcriptase polymerase chain reaction from human placental cells. The BMP-4/7 fusion gene was then generated through recombination. Rabbit BMSCs were transfected with the recombinant AAV vectors carrying AAV-BMP-4/7 with multiplicity of infection values. Cell growth curves were drawn to evaluate the biological effects of AAV-BMP-4/7 on cell activity. The transfection efficiency was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The ossification of cells was evaluated by observing alkaline phosphatase (ALP) and osteocalcin (OC) activity after transfection for 7 and 14 days. The cells were then transfected with AAV-BMP-4/7 and AAV-enhanced green fluorescent protein. We successfully constructed the recombinant adeno-associated virus with the BMP-4/7 fusion gene. The transfection efficiency of AAV-BMP-4/7 was approximately 72% without significant biological effects on cell activity. Cell ossification was significant after transfection with AAV-BMP-4/7. The 1 x 10(5) vg/cell multiplicity of infection value of transfection efficiency was more than 5 x 10(4) vg/cell (59.38%). Significantly higher ALP and OC activity occurred in the AAV-BMP-4/7 transfection groups than in the AAV-enhanced green fluorescent protein groups (t(ALP) = 896.88, P < 0.001; t(OC) = 543.24, P < 0.01). The AAV-BMP-4/7 fusion gene can highly efficient transfect rabbit BMSCs cultured in vitro and it has significant ossification activity.

Detection of PAX3/PAX7-FKHR fusion transcripts in rhabdomyosarcoma and other small round cell tumors by 1-step reverse transcriptase polymerase chain reaction: a novel tool for diagnosis and differentiation

Rhabdomyosarcoma (RMS) is one of the most frequent soft tissue sarcomas in children. It is sometimes difficult to distinguish it from other small round cell tumors (SRCTs) depending on microscopic observations; although their treatment and prognosis varied widely, the same happens between alveolar RMS (ARMS) and embryonal RMS (ERMS). The role of PAX3/PAX7-FKHR fusion gene has been reported in ARMS but not in ERMS and SRCT. The aim of this study was to explore its value in RMS diagnosis and differentiation. Ninety-eight patients with ARMS (n = 13), ERMS (n = 25), pleomorphic RMS (n = 5), Ewing sarcoma (n = 11), neuroblastoma (n = 18), lymphoma (n = 24), and uncertain SRCT (n = 2) were analyzed. One hundred fifteen RNA samples were extracted from the primary tumor tissue at initial presentation and relapse. One-step reverse transcriptase polymerase chain reaction assays for the PAX3/PAX7-FKHR fusion transcripts were performed. Molecular findings were compared with original histologic diagnoses. PAX3-FKHR fusion transcript was detected in 9 ARMS samples, PAX7-FKHR fusion transcript was detected in 7 ARMS samples, and 2 uncertain SRCTs were detected; none of them were detected in ERMS, Ewing sarcoma, neuroblastoma, and lymphoma. Direct sequencing of PAX3 coding regions revealed a heterozygous mutation A→G (nt1380) at codon 448 (AAT→GAT), resulting in substitution of Asn-448 for Asp. Detection of PAX3/PAX7-FKHR fusion transcripts by 1-step reverse transcriptase polymerase chain reaction is a novel tool for RMS diagnosis and differentiation.

Detection rates of the swine torque teno viruses (TTVs), porcine circovirus type 2 (PCV2) and hepatitis E virus (HEV) in the livers of pigs with hepatitis

Porcine circovirus type 2 (PCV2) and hepatitis E virus (HEV) are the most recently recognized causes of infectious hepatitis of pigs and may or may not act independently in the development of the disease. Recently it has been suggested that swine torque teno viruses (TTVs), in co-infections with some swine viral pathogens, may potentiate the severity of disease. In order to search for virological cofactors associated with infectious hepatitis in pigs, we investigated the liver tissues, to determine the presence of TTVs, PCV2 and HEV of naturally infected pigs and analysed the prevalence of both genogroups of the TTVs in the hepatitis lesions. Histopathological techniques, nested-polymerase chain reactions (nPCRs), polymerase chain reaction (PCR) and one-step reverse transcriptase polymerase chain reaction (RT-PCR) were applied to detect hepatitis lesions, TTVs genogroups 1 and 2, PCV2 and HEV infection. Of the livers examined 58% (29/50) had mild to moderate hepatitis and 74% (37/50), 56% (28/50) and 26% (13/50) samples were nPCR, PCR and RT-PCR positive for TTVs PCV2 and HEV respectively. TTVs were detected in 84% (16/19) of the samples which were determined to be of mild severity while present in almost all (90% or 9/10) samples identified as having moderate hepatitis lesions. Additionally, the livers of 12 out of 21 (57%) pigs without the hepatitis lesions were positive for TTVs. These results demonstrate an association between TTVs and infectious hepatitis of pigs in concomitant infections with PCV2 and/or HEV and indicated that TTVs may play a role as a cofactor in the pathogenesis of disease.

One-step reverse transcriptase polymerase chain reaction for the diagnosis of respiratory syncytial virus in children

Human respiratory syncytial virus (HRSV) is the main cause of acute lower respiratory tract infections in infants and children. Rapid diagnosis is required to permit appropriate care and treatment and to avoid unnecessary antibiotic use. Reverse transcriptase (RT-PCR) and indirect immunofluorescence assay (IFA) methods have been considered important tools for virus detection due to their high sensitivity and specificity. In order to maximize use-simplicity and minimize the risk of sample cross-contamination inherent in two-step techniques, a RT-PCR method using only a single tube to detect HRSV in clinical samples was developed. Nasopharyngeal aspirates from 226 patients with acute respiratory illness, ranging from infants to 5 years old, were collected at the University Hospital of the University of Sao Paulo (HU-USP), and tested using IFA, one-step RT-PCR, and semi-nested RT-PCR. One hundred and two (45.1%) samples were positive by at least one of the three methods, and 75 (33.2%) were positive by all methods: 92 (40.7%) were positive by one-step RT-PCR, 84 (37.2%) by IFA, and 96 (42.5%) by the semi-nested RT-PCR technique. One-step RT-PCR was shown to be fast, sensitive, and specific for RSV diagnosis, without the added inconvenience and risk of false positive results associated with semi-nested PCR. The combined use of these two methods enhances HRSV detection.

An integrated microfluidic system using magnetic beads for virus detection

An integrated system capable of sample pretreatment using antibody-conjugated magnetic beads and one-step reverse transcriptase–polymerase chain reaction (RT-PCR) on a microfluidic system was developed to accelerate the detection of RNA viruses such as dengue virus or enterovirus 71. The targeted virus in the sample was first captured by the specific antibody-conjugated magnetic beads, which were manipulated by micro-electromagnets made of micro-electro-mechanical systems technology. The RNA of the targeted virus then underwent thermal lysis and was reverse-transcripted to cDNA using a μRT-PCR module. The sensitivity to detect dengue virus is around 10–100 PFU, which is equivalent to the commercial RNA extraction kit and a large-scale RT-PCR machine. This microsystem can specifically detect 4 serotypes of dengue virus, as well as enterovirus 71. The specificity was warranted by both antibody and primer. The microfluidic system allows automatic process of sample including mixing, incubation, and reaction. The antibody-conjugated magnetic beads offer sample pretreatment of purification and concentration. The integration of antibody-conjugated magnetic beads into the microfluidic system is promising for fast molecular diagnosis of microorganisms.

Chronic consumption of a low-fat diet leads to increased hypothalamic agouti-related protein and reduced leptin

Objective This study examined the hypothesis that dietary fat under ad libitum feeding conditions influences expression levels (mRNA) of the mouse agouti-related protein (AgRP), leptin, leptin receptor (OBRb), and neuropeptide Y (NPY) at early stages of development. Methods C57Bl/6J male mice were placed on a high-fat diet (HFD) or a low-fat diet (LFD) shortly after weaning. Groups of mice were euthanized at various ages and real-time one-step reverse transcriptase polymerase chain reaction was used to analyze gene expression in the hypothalamus (AgRP, NPY, OBRb), the adrenal gland (AgRP), the testis (AgRP), and epididymal fat (leptin). Results Leptin expression increased linearly with age but only under the HFD despite body weight gain under both diets. This pattern of expression coincided with reduced expression of hypothalamic AgRP under an HFD, whereas OBRb and NPY did not fluctuate in response to diet. By contrast, consumption of an LFD (i.e., high carbohydrate) increased hypothalamic AgRP and suppressed adipose leptin, which is consistent with the notion that leptin could regulate AgRP centrally. In contrast, AgRP expression in the adrenal gland initially decreased and then increased with age under both diets. Conclusions Dietary fat can have a tissue-dependent effect on AgRP that may be unfettered by leptin under an HFD.

A single nucleotide mutation in Nppc is associated with a long bone abnormality in lbab mice

BackgroundThe long bone abnormality (lbab) mouse is a new autosomal recessive mutant characterized by overall smaller body size with proportionate dwarfing of all organs and shorter long bones. Previous linkage analysis has located the lbab mutation on chromosome 1 between the markers D1Mit9 and D1Mit488.ResultsA genome-based positional approach was used to identify a mutation associated with lbab disease. A total of 122 genes and expressed sequence tags at the lbab region were screened for possible mutation by using genomic DNA from lbabl/lbab, lbab/+, and +/+ B6 mice and high throughput temperature gradient capillary electrophoresis. A sequence difference was identified in one of the amplicons of gene Nppc between lbab/lbab and +/+ mice. One-step reverse transcriptase polymerase chain reaction was performed to validate the difference of Nppc in different types of mice at the mRNA level. The mutation of Nppc was unique in lbab/lbab mice among multiple mouse inbred strains. The mutation of Nppc is co-segregated with lbab disease in 200 progenies produced from heterozygous lbab/+ parents.ConclusionA single nucleotide mutation of Nppc is associated with dwarfism in lbab/lbab mice. Current genome information and technology allow us to efficiently identify single nucleotide mutations from roughly mapped disease loci. The lbab mouse is a useful model for hereditary human achondroplasia.

Effects of p, p′-dichlorodiphenyldichloroethylene on the expressions of transferrin and androgen-binding protein in rat Sertoli cells

The mechanisms of reproductive malfunction of male mammals caused by 2,2-bis(4-chlorophenyl)-1,1-dichloroethylene ( p, p′-DDE, hereafter DDE) remain unknown. To explore the effects of DDE on the expressions of transferrin (Tf) and androgen-binding protein (ABP), we isolated Sertoli cells from healthy immature rats (18–20 days SD rats), set up Sertoli cell cultures, evaluated the toxicity, and measured the expression levels of mRNA of Tf and ABP genes by the one-step reverse transcriptase polymerase chain reaction method after cultured Sertoli cells were in vitro exposed to DDE at different concentrations for 24 h. The results showed that the number and survival rate of Sertoli cells decreased sharply with increased doses of DDE. The expression level of Tf mRNA decreased, whereas ABP mRNA increased gradually with increased DDE doses. There existed an obvious dose–effect relationship between the concentration of DDE and the expression levels of Tf mRNA and ABP mRNA. These findings suggest that DDE may inhibit the expression of Tf and up-modulate expression of ABP in cultured rat Sertoli cells.

Upregulation of the BRCA1 gene in human germ cells and in preimplantation embryos

The quantification of BRCA1 messenger RNA molecules by a quantitative competitive one-step reverse transcriptase polymerase chain reaction method indicates that BRCA1 is upregulated both in human male and female germ cells and in preimplantation embryos. Because BRCA1 is involved in several pathways that participate in preserving intact chromosome and genome integrity, these data suggest that BRCA1 dysfunction might alter human embryogenesis or fertility.

Detection of SYT-SSX Rearrangements in Synovial Sarcomas by Real-Time One-Step RT-PCR

Synovial sarcomas are aggressive tumors of adolescent and young adults that account for up to 10% of soft tissue sarcomas. Cytogenetically, they are characterized by translocation t(X;18), which is found in more than 95% of tumors. In most cases, it results in fusion of the SYT gene with the SSX1 or SSX2 gene, thus creating SYT-SSX1 or SYT-SSX2 rearrangement. The 2 types of gene fusion have been correlated with histologic variants and prognosis of synovial sarcomas. In this study, we developed a simple and rapid method for the simultaneous detection of SYT-SSX1 and SYT-SSX2 rearrangements by using a LightCycler real-time one-step reverse transcriptase polymerase chain reaction (RT-PCR) technology (Roche). Oligonucleotide probes were designed so that the donor probe would span a fusion point and the acceptor probe would be complementary to the SSX1 sequence but have 2 nucleotide mismatches with SSX2 sequence. Such a design allows simultaneous amplification of 2 types of rearrangement in the same reaction but distinguishes them based on differences in melting temperature detected by melting curve analysis after PCR. With this method, 27 tumors (9 synovial sarcomas and 18 nonsynovial sarcomas) were studied and showed SYT-SSX1 rearrangement in 6 cases and SYT-SSX2 in 3 cases. These results had complete correlation with the finding of conventional RT-PCR and direct sequencing. In conclusion, we have developed a fast, accurate, and simple method for the detection of 2 major types of SYT-SSX rearrangement by using LightCycler RT-PCR and melting curve analysis.