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PubMed2001

Identification of canine coronavirus strains from feces by S gene nested PCR and molecular characterization of a new Australian isolate.

A nested PCR (nPCR) assay for the detection of canine coronavirus (CCV) in fecal samples is described. The target sequence for the assay was a 514-bp fragment within the spike (S) glycoprotein gene. The sensitivity of the assay is extremely high, detecting as little as 25 50% tissue culture infective doses per g of unprocessed feces. A clinical trial using dogs challenged orally with CCV SA4 and CCV NVSL was used to compare viral isolation and the nPCR assay as detection techniques over a 2-week period of infection. Virus isolation detected CCV shedding from day 4 to 9 postchallenge, while the nPCR assay detected CCV shedding from day 4 to 13 postchallenge. Cloning and sequencing of the nPCR assay product enabled investigation of the evolutionary relationships between strains within the S gene. The simple and rapid procedure described here makes this assay an ideal alternative technique to electron microscopy and viral isolation in cell culture for detection of CCV shedding in feces. The described assay also provides a method of identifying new strains of CCV without the complicated and time-consuming practice of raising antibodies to individual strains. This is illustrated by the identification, for the first time, of an Australian isolate of CCV (UWSMN-1).

Amino Acid Sequence↗
PubMed2005

Comparison of five DNA extraction methods and optimization of a b1 gene nested PCR (nPCR) for detection of Toxoplasma gondii tissue cyst in mouse brain.

Toxoplasmosis, caused by Toxoplasma gondii, is an important parasitic disease worldwide. Different techniques have been developed for T. gondii detection. At present, polymerase chain reaction (PCR) has been widely used. However, PCR for identifying T. gondii remains unsatisfactory in many laboratories because of lack of standardization and variations in efficiency. In the present study, we optimized a nested PCR protocol (n-PCR) in order to compare the amplification of T. gondii DNA, after being extracted from mouse brain by five different DNA extraction methods including phenol chloroform, QIAamp DNA minikit, Genomic DNA purification kit and Chelex with or without proteinase K. All DNA extraction methods were able to extract DNA from a single tissue cyst from mouse brain. However, among the five DNA extraction methods, the Chelex without proteinase K appeared to be the most rapid and easiest.

Animals↗
PubMed2003

Germline cell death is inhibited by P-element insertions disrupting the dcp-1/pita nested gene pair in Drosophila.

Germline cell death in Drosophila oogenesis is controlled by distinct signals. The death of nurse cells in late oogenesis is developmentally regulated, whereas the death of egg chambers during mid-oogenesis is induced by environmental stress or developmental abnormalities. P-element insertions in the caspase gene dcp-1 disrupt both dcp-1 and the outlying gene, pita, leading to lethality and defective nurse cell death in late oogenesis. By isolating single mutations in the two genes, we have found that the loss of both genes contributes to this ovary phenotype. Mutants of pita, which encodes a C2H2 zinc-finger protein, are homozygous lethal and show dumpless egg chambers and premature nurse cell death in germline clones. Early nurse cell death is not observed in the dcp-1/pita double mutants, suggesting that dcp-1+ activity is required for the mid-oogenesis cell death seen in pita mutants. dcp-1 mutants are viable and nurse cell death in late oogenesis occurs normally. However, starvation-induced germline cell death during mid-oogenesis is blocked, leading to a reduction and inappropriate nuclear localization of the active caspase Drice. These findings suggest that the combinatorial loss of pita and dcp-1 leads to the increased survival of abnormal egg chambers in mutants bearing the P-element alleles and that dcp-1 is essential for cell death during mid-oogenesis.

Amino Acid Sequence↗
PubMed1994

[Detection of Mycobacterium tuberculosis using a nested-primer gene amplification assay].

A nested-primer gene amplification assay (NPGAA) was established by using two pairs of primers, an outside pair of primers and an inside pair of primers, sequences of which were from M. tuberculosis(Mtb) gro EL genes. The outside pair of primers should amplify a 576-bp piece of the Mtb gro EL gene that contains sites for the inside pair of primers, which should amplify a 344-bp piece. The results shows NPGAA'S detectable limitation was 1 organism/ml and no amplification products were produced from DNAs of other mycobacterium species tested in this study. For detecting Mtb, the entire NPGAA, from sample preparation to data analysis, can be completed within 6-8 hours. When identifying AFB cultures isolated from lesions of skin diseases, the same positive size patterns were obtained.

Base Sequence↗
PubMed2001

Detection of Borrelia duttonii, a tick-borne relapsing fever agent in central Tanzania, within ticks by flagellin gene-based nested polymerase chain reaction.

Argasid ticks collected in the site near Mvumi Hospital, Dodoma, Tanzania, were subjected to flagellin gene-based nested polymerase chain reaction (PCR) amplification for examination of borrelial infections. Eight of 13 ticks gave a strong 350-bp signal; three had a weak signal at the same size, and the rest were negative. Sequence determination of eight of the positive samples resulted in three types of flagellin gene sequences. The first type of sequence (shown by three individuals) was identical to that of Borrelia duttonii strain Ly. The second type of sequence from PCR products from four individual ticks had only one base substitution without amino acid alteration in deduced protein sequence. The third type of sequence was different from that of any other Old World relapsing fever borreliae, and the tick was thought to be infected with an unknown Borrelia species. Ticks were also examined to determine the nucleotide sequence of the mitochondrial 16S rRNA gene. The partial sequence of approximately 470 bases was aligned for comparison with previously published sequences to identify the species. The sequences of 13 individual ticks were all identical, and the sequence similarity analysis revealed the ticks should be classified as members of the Ornithodoros porcinus species. The PCR method described in this report appears to be a reliable tool for the detection of borreliae and epidemiological study of tick-borne relapsing fever.

Amino Acid Sequence↗
PubMed1992

[Rapid detection of mecA gene by nested PCR for diagnosis of methicillin resistance in Staphylococcus aureus].

In recent years, the most common causative organism of hospital infections has been methicillin resistant Staphylococcus aureus (MRSA). The major mechanism of beta-lactam resistance in MRSA is attributed to the production of a specific penicillin binding protein (PBP2'), which is a product of mecA gene, with extremely low binding affinities to beta-lactams. In the present study, we have established a rapid identification method of MRSA by sensitive detection of mecA gene using nested PCR. Nested PCR method amplifying the target DNA in two steps enhanced the efficiency of the second round amplification. By means of this method, mecA gene was successfully detected in clinical samples, such as blood, pus, sputum and feces within 3-4 hrs. Rapid diagnosis of MRSA-bacteremia is particularly important for prevention of sever systemic infection. There are some strains of S. aureus which possess mecA gene in spite of low minimal inhibitory concentration of DMPPC. In these strains expression of mecA gene is induced by contact of beta-lactams and they obtain methicillin resistance. Using nested PCR method, these latent MRSA are rapidly and certainly detectable. This method should be useful for early and effective detection of MRSA hospital infections.

Cross Infection↗
PubMed1996

The alcohol dehydrogenase gene is nested in the outspread locus of Drosophila melanogaster.

This report describes the structure and expression of the outspread (osp) gene of Drosophila melanogaster. Previous work showed that chromosomal breakpoints associated with mutations of the osp locus map to both sides of the alcohol dehydrogenase gene (Adh), suggesting that Adh and the adjacent gene Adhr are nested in osp. We extended a chromosomal walk and mapped additional osp mutations to define the maximum molecular limit of osp as 119 kb. We identified a 6-kb transcript that hybridizes to osp region DNA and is altered or absent in osp mutants. Accumulation of this RNA peaks during embryonic and pupal periods. The osp cDNAs comprise two distinct classes based on alternative splicing patterns. The 5' end of the longest cDNA was extended by PCR amplification. When hybridized to the osp walk, the 5' extension verifies that Adh and Adhr are nested in osp and shows that osp has a transcription unit of > or = 74 kb. In situ hybridization shows that osp is expressed both maternally and zygotically. In the ovary, osp is transcribed in nurse cells and localized in the oocyte. In embryos, expression is most abundant in the developing visceral and somatic musculature.

Alcohol Dehydrogenase↗
PubMed1990

Rapid and sensitive detection of Mycobacterium leprae using a nested-primer gene amplification assay.

By using a set of four nested oligonucleotide primers, a two-step polymerase chain reaction assay for the detection and identification of Mycobacterium leprae that does not require the use of radioactivity labeled hybridization probes was developed. The nested-primer procedure amplified a 347-base-pair product from M. leprae genomic DNA. No amplification products were produced from DNAs of 19 other Mycobacterium species, 19 non-Mycobacterium species, mouse cells, or human cells. Minor amplification products were observed with three additional Mycobacterium species, i.e., "M. lufu", M. simiae, and M. smegmatis. These products were easily distinguished from the M. leprae product by size and restriction enzyme cleavage patterns. The assay could amplify the 347-base-pair product from samples containing as little as 3 fg of M. leprae genomic DNA--the amount of DNA in a single bacillus. The assay also amplified target sequences in crude lysates of M. leprae bacilli isolated from tissue biopsy specimens from infected animals and humans. The entire assay, from sample preparation to data analysis, can be completed in less than 8 h.

Animals↗
PubMed2007

Papillary thyroid cancer and polymorphic variants in TSHR- and RET-related genes: a nested case-control study within a cohort of U.S. radiologic technologists.

Several variants in the TSHR and RET signaling pathways genes have been reported to be related to cancer risk. We hypothesized that polymorphic variants in these genes are associated with the risk of papillary thyroid cancer. A nested case-control study was conducted within the U.S. Radiologic Technologists cohort. Eligible validated papillary thyroid cancer cases (n = 167) and frequency-matched (by sex and birth year) controls (n = 491) donated blood for analysis. There were no statistically significant associations between papillary thyroid cancer and 10 selected polymorphic variants in analyses of men and women combined. A borderline significant increasing risk was found for RET G691S (P(trend) = 0.05) and was especially pronounced among young women. For women under 38 years (the median age at diagnosis), the odds ratios were 2.1 (95% confidence interval, 1.2-3.7) for those heterozygous for the RET G691S polymorphism and 3.7 (95% confidence interval, 1.1-11.8) for those who were homozygous (P(trend) = 0.001). Our data provide limited evidence that TSHR- and RET-related genes are related to papillary thyroid cancer risk.

Adult↗
PubMed2005

Amplification of P1 and 16S rRNA genes by nested PCR for detection of Mycoplasma pneumoniae in paediatric patients.

Mycoplasma (M.) pneumoniae is the most frequent atypical pathogen responsible for community-acquired respiratory infection in children and adults. The etiologic diagnosis of these infections still remains difficult. This is mainly due to the absence of characteristic clinical findings, and to the available detection methods (serology and culture) which are time consuming, insensitive and non-specific. To improve the detection of this infectious agent, nested polymerase chain reaction (PCR) analysis was developed. A total of 46 nasal aspirates, from children hospitalised with severe lower respiratory tract infection and in whom M. pneumoniae was suspected, were analysed for the presence of M. pneumoniae DNA by PCR. Routine microbiological investigations revealed no virus in these 46 samples. Using nested PCR, two targets were amplified: the sequences of 16S ribosomal (r) RNA gene (rDNA) and P1 adhesin gene. Evidence of M. pneumoniae infection was identified in four paediatric patients. The amplification of 16S rDNA was found to be more sensitive for the detection of M. pneumoniae. Our results suggest that amplification of the 16S rDNA by nested PCR and detection of the amplification products by visual inspection of the polyacrylamide gel should allow the rapid diagnosis of M. pneumoniae in respiratory tract infection in paediatric patients.

Adhesins, Bacterial↗
PubMed2002

The DSC1 channel, encoded by the smi60E locus, contributes to odor-guided behavior in Drosophila melanogaster.

Previously, we generated P-element insert lines in Drosophila melanogaster with impaired olfactory behavior. One of these smell-impaired (smi) mutants, smi60E, contains a P[lArB] transposon in the second intron of the dsc1 gene near a nested gene encoding the L41 ribosomal protein. The dsc1 gene encodes an ion channel of unknown function homologous to the paralytic (para) sodium channel, which mediates neuronal excitability. Complementation tests between the smi60E mutant and several EP insert lines map the smell-impaired phenotype to the P[lArB] insertion site. Wild-type behavior is restored upon P-element excision. Evidence that reduction in DSC1 rather than in L41 expression is responsible for the smell-impaired phenotype comes from a phenotypic revertant in which imprecise P-element excision restores the DSC1 message while further reducing L41 expression. Behavioral assays show that a threefold decrease in DSC1 mRNA is accompanied by a threefold shift in the dose response for avoidance of the repellent odorant, benzaldehyde, toward higher odorant concentrations. In situ hybridization reveals widespread expression of the dsc1 gene in the major olfactory organs, the third antennal segment and the maxillary palps, and in the CNS. These results indicate that the DSC1 channel contributes to processing of olfactory information during the olfactory avoidance response.

Animals↗
PubMed1991

Cytomegalovirus DNA detection of an immediate early protein gene with nested primer oligonucleotides.

A rapid and sensitive polymerase chain reaction (PCR) was developed to detect conserved sequences from the immediate early gene of human cytomegalovirus (HCMV). The primers sequences were from EcoRI J fragment of Ad169. The first primer set was selected to amplify a 242 bp fragment and the next primer set was nested within the first and amplified a 146 bp fragment. With the single PCR system it was possible to detect 100 fg HCMV DNA but with double PCR 5-10 fg were detectable. Specific amplification was seen in urines from patients with HCMV infections. 20 urine samples were analysed by single PCR, double PCR and virus cultivation. The double PCR was the most sensitive method. Urines from healthy seropositive persons and cells infected with other members of the herpes virus family were negative with all three methods. This suggests that specific amplification by double PCR is sensitive and can be used for rapid detection of HCMV DNA in cases with activated infection.

Antigens, Viral↗
PubMed1993

Sequence analysis of the phage 21 genes for prohead assembly and head completion.

Phage 21 is a temperate lambdoid coliphage, and its head-encoding genes, as well as those of phage lambda, are descended from a common ancestral phage. The head protein-encoding genes of phage 21 have been sequenced, confirming earlier genetic studies indicating that the head-encoding genes of 21 and lambda are analogous in location, size, and function. The phage 21 head-encoding genes identified (and their lambda analogues) include: 3(W), 4(B), 5(C), 6(Nu3), shp (D), 7(E), and 8(FII), respectively. An open reading frame, orf1, is analogous in position and shares some sequence identity with FI, a phage lambda gene involved in DNA packaging. The phage 21 major head protein, gp7, is predicted to have strong sequence identity (65%) with the lambda major capsid protein, gpE, including amino acids known to be important for capsid form determination. The nested genes 5/6 of phage 21 and C/Nu3 of lambda differ by several rearrangements including deletions and a triplication. The possibility that lambda genes C/Nu3 evolved from ancestal nested genes containing a triplication is discussed.

Amino Acid Sequence↗
PubMed1995

[Amplifying Helicobacter pylori's urease gene by nested polymerase chain reaction].

By using nested polymerase chain reaction (PCR) composed of three primers that derived from Helicobacter pylori's (Hp) urease gene region, we have not only successfully amplified control Hp and 214 clinic samples but also studied deeply preparation of the sample, composition of PCR reaction liquid and circulatory mental conditions of the PCR. So this method shortens the operation time, simplifies processes and reduces costs. Compared with urease test (54%) and Warthin-Starry silver stained test (50%), ever used the positive rate of PCR is the highest (61%), so our method is more specific, sensitive, simple, rapid and accurate, and could be applied to the routine clinical detection.

Adolescent↗
PubMed1998

Simultaneous detection of five mutations in the steroid 21-hydroxylase gene using nested allele-specific amplification.

Congenital adrenal hyperplasia due to deficiency of steroid 21-hydroxylase (CYP21) is most frequently due to mutations that arise from the nearby CYP21 pseudogene. The mechanism involves either unequal crossing over, which deletes part of the CYP21 functional gene, or gene conversion which puts a mutation from the pseudogene into the functional gene. We have devised an assay to rapidly screen for five known mutations that are due to gene conversion within an 1,800 bp region of the CYP21 gene--I172N, V281L, Q318X, R356W, and a cluster of mutations in exon 6 (I236N, V237E, M239K). This method is based on a set of nested allele-specific polymerase chain reactions done simultaneously in one tube, for which we suggest the acronym NASA, for nested allele-specific amplification. The assay is capable of detecting the mutations individually as well as all combinations of mutations tested.

Adrenal Hyperplasia, Congenital↗
PubMed1995

Detection of measles virus genome directly from clinical samples by reverse transcriptase-polymerase chain reaction and genetic variability.

A simple and sensitive method for the detection of measles virus genome was developed, amplifying the regions encoding the nucleocapsid (N) protein and hemagglutinin (H) protein of measles virus by reverse transcriptase-polymerase chain reaction (RT-PCR). We examined a variety of measles patients: 28 patients with natural infection, 4 with measles encephalitis and 1 with subacute sclerosing panencephalitis (SSPE). In 28 patients with natural measles infection a single step PCR amplifying the N region resulted in a high detection rate for all plasma samples (28/28) within 3 days of the onset of rash and 80% (20/25) even on day 7 of the onset of rash and later. Within 3 days of the onset of rash, 24/25 (96.0%) of nasopharyngeal secretions (NPS) and 27/28 (96.4%) of peripheral blood mononuclear cells (PBMC) were positive for the N region PCR and the positivity rate of PCR decreased in NPS and PBMC after 7 days of the rash. In acute measles infection, measles genome was detected in all cell fractions, CD4, CD8, B cells, and monocytes/macrophages by the H gene nested PCR. Measles genome was also detected from cerebrospinal fluids (CSF) in patients with measles encephalitis, SSPE, and acute measles by the H gene nested PCR. PCR products of the N and H regions were sequenced and we confirmed the presence of measles genome. Based on the sequence data, chronological sequence differences were observed over the past 10 years. The sequences obtained from the SSPE patient were closely related to those of the wild viruses that were circulating at the time when the patient initially acquired measles. RT-PCR for NPS, PBMC, CSF, and plasma provides a useful method for the diagnosis of measles and molecular epidemiological study in addition to virus isolation.

Adolescent↗
PubMed2000

Endogenous ecotropic murine leukemia viral (MuLV) envelope protein as a new autoantigen reactive with non-obese diabetic mice sera.

The identification and characterization of autoantigens associated with autoimmune IDDM (insulin dependent diabetes mellitus) would help to elucidate the pathogenic mechanism of this disease as well as to design antigen-based immunotherapy. Non-obese diabetic (NOD) mice have been used as the best model for studying the pathogenesis of human IDDM. To identify new autoantigens associated with IDDM, the lambda gt11-cDNA library from MIN6N8a, NOD-derived pancreatic beta cell line, was constructed and then candidate autoantigen clones were screened with prediabetic NOD sera. Nine positive clones were selected from 2x10(5)phage plaques. The nucleotide sequencing and homology searching showed that six of the nine positive clones had part of the endogenous ecotropic murine leukemia viral (MuLV) envelope gene. Nested deletion of this envelope gene revealed that the leucine zipper region in the transmembrane domain of MuLV envelope protein was the target epitope(s) reactive with prediabetic NOD mice sera. The prevalence of MuLV envelope protein-positive antibody in NOD mice was around 46%, while the non-NOD mice strains including BALB/c, ICR, C57BL/6, and SJL/J mice did not produce this envelope protein-reactive antibody. The expression of endogenous ecotropic MuLV envelope gene in NOD mouse pancreas was distinct in those with severe insulitis. However, both prediabetic and diabetic NOD mice did not show the MHC class II-restrictive cellular autoimmunity against our purified recombinant envelope protein. In this study, we showed that the endogenous ecotropic MuLV envelope protein was a new autoantigen reactive with the activated NOD humoral immune system.

Animals↗