Polymerase Chain Reaction Procedure Research Articles

Molecular diagnosis of 5α-reductase-2 gene mutation in two Indian families with male pseudohermaphroditism

To identify the genotype of two Indians with male pseudohermaphroditism. Standard radioimmunoassay procedure was used for estimating hormonal levels. Conventional cytogenetic analysis was carried out for diagnosing the genetic sex in these subjects with genital ambiguity. Molecular analysis was carried out by standard polymerase chain reaction procedure using different sets of primers and reaction conditions specific for the 5alpha-reductase type 2 gene (SRD5A2) gene. Direct sequencing was carried out using the ABI Prism dye terminator sequencing kit and the ABI 310 sequencing apparatus. We found an SRD5A2 gene mutation in exon 5, where arginine is substituted with glutamine (R246Q), in two males with pseudohermaphroditism and ambiguous genitalia from unrelated families. This is the first time this mutation has been reported in individuals from India. Identification of the R246Q mutation of the SRD5A2 gene from two unrelated Indian families possibly extends the founder gene effect.

Sex determination in goat by amplification of the HMG box using duplex PCR

The objective of this study was to obtain a fast, accurate and reliable method of determining the sex of goat embryos prior to implantation through amplification of the high-motility-group (HMG) box of the sex-determining region of the Y chromosome (SRY) gene of the goats. Goat specific primers were designed for duplex polymerase chain reaction (PCR). As an internal control gene, the goat beta-action gene sequence was simultaneously amplified together with the HMG box of goat SRY gene. Males showed both 1 SRY band and 1 beta-action band, but only 1 beta-action band was present in the agarose gel electrophoresis of females. The result indicated that the goat HMG-box sequence motif of SRY was male specific. Afterward, the optimized PCR procedure was applied in 30 embryo biopsies and the biopsied embryos were transferred into 30 recipient female goats. The sex of the 13 kids proved anatomically corresponded to the sex determined by PCR (100% accuracy). Thus, this study showed that this duplex PCR method can be applied to sex the goat pre-implantation embryos and to manipulate the sex ratio of offspring in goat breeding programs.

DNA-labelled cytidine assay for the quantification of CAG repeats

The sequencing procedure has been used to determine the size of the CAG repeat expansion for the diagnosis of genetic disorders. Likewise, standard polymerase chain reaction (PCR) and gel electrophoresis techniques are applied for screening large number of patients. The trinucleotide repeats (TNR) region amplification by means of the PCR procedure was initially performed using 32-P end-labelled primers and currently carried out with fluorescently end-labelled primers. The goal to obtain reliable TNR quantification assays, at low cost and short assay times, represents a challenge for the molecular diagnosis aimed at massive screening of affected populations. In the current work, we obtained preliminary results of a new methodology for the detection and size estimation of CAG expanded alleles. The assay was based on an indirect enzyme linked immunosorbent assay (ELISA) for quantifying the amount of labelled cytidines in DNA molecules. The label, 6-( p-bromobenzamido)caproyl radical, was introduced by the transamination and acylation reactions. A group of model sequences containing different numbers of CAG repeats, as well as the ATXN3 (ataxin 3) gene (from subjects suffering type 3 spinocerebellar ataxia SCA3) were used for assay standardization. The assay is simple, inexpensive, and easy to perform and differentiates distinct degrees of CAG expansions.

Preimplantation Genetic Diagnosis: Rationale and Ethics, an Islamic Perspective

DOI: http://dx.doi.org/10.5915/39-4-6316 Preimplantation genetic diagnosis (PGD) is a relatively new procedure meant to diagnose genetic or chromosomal defects in fertilized eggs produced by in vitro fertilization (IVF) so as to avoid implanting an embryo. It has also been used to diagnose mutations associated with several types of cancer. One or two blastomeres are removed from the preembryo (8-16 cell morula stage). A polymerase chain reaction (PCR) procedure tests for genetic mutations, and fluoresence in situ hybridization (FISH) tests for chromosomal abnormalities. The various clinical applications of PGD are presented and classified into acceptable, questionable, and unacceptable. The main advantage of PGD is that it will eliminate or significantly reduce the risk couples with genetic diseases face of having a baby with those diseases, thus avoiding some terminations of pregnancy. However, PGD raises significant ethical issues, the most important of which is the sanctity of human life. While PGD does not result in loss of biopsied healthy preembryos, it involves discarding of affected human embryos. The arguments for and against this are discussed in detail. Further, the implications of PGD on a societal level and the danger of using it for eugenics are also discussed. While the use of PGD for sex selection for medical reasons is acceptable, its use for nonmedical reasons is controversial. Islam encourages scientific developments as long as they benefit humankind and do not contradict basic Islamic rulings. Most Muslim scholars approve of PGD use as it involves a preembryo before it is implanted and allows for benefits to the couple involved, i.e. the prevention of having a baby with genetic diseases. They do not approve of its use for sex selection for nonmedical reasons.

Interrelationships Between “<I>Candidatus</I> Phytoplasma asteris” and Its Leafhopper Vectors (Homoptera: Cicadellidae)

The titer of chrysanthemum yellows phytoplasma (CYP, "Candidatus Phytoplasma asteris") in the three vector species Euscelis incisus Kirschbaum, Euscelidius variegatus Kirschbaum, and Macrosteles quadripunctulatus Kirschbaum (Homoptera: Cicadellidae) was measured after controlled acquisition from infected Chrysanthemum carinatum (Schousboe) (daisy) plants. Phytoplasma DNA was quantified in relation to insect DNA (genome units [GU] of phytoplasma DNA per ng of insect DNA) by using a quantitative real-time polymerase chain reaction (PCR) procedure. The increase in phytoplasma titer recorded in hoppers after they were transferred to plants that were nonhosts for CYP provides definitive evidence for phytoplasma multiplication in leafhoppers. CYP multiplication over time in M. quadripunctulatus was much faster than in E. incisus and E. variegatus. CYP titer was also highest in M. quadripunctulatus, and this was reflected in the latent period in the insect. The mean latent period of CYP in M. quadripunctulatus was 18 d versus 30 d in E. variegatus. M. quadripunctulatus was the most efficient vector, giving 100% transmission for single insects compared with 75-82% for E. incisus or E. variegatus, respectively. By sequential transmission, we analyzed the time course of transmission: E. variegatus were persistently infective for life or until shortly before death. Occasionally, leafhoppers failed to maintain continuity of infectivity even after completion of the latent period. PCR analysis of transmitter and nontransmitter E. variegatus adults showed that some nontransmitters were CYP positive, whereas others were CYP negative. These findings suggest that both midgut and salivary gland barriers play a role in transmission efficiency.

Small-pool PCR analysis of microsatellite instability in HNPCC

The contributions and limitations of the traditional method for evaluating microsatellite instability by standard polymerase chain reaction (PCR) in identifying mismatch repair (MMR) gene mutations in the Lynch syndrome form of hereditary nonpolyposis colon cancer (HNPCC) are discussed. A new form of HNPCC (familial colon cancer type X or FCCX), in which deleterious mutations in major MMR genes and microsatellite instability have not been identified, was viewed as possibly having attenuating mutations in MMR genes as its genetic basis. A highly sensitive and quantitative method of microsatellite instability analysis, small-pool PCR, is needed to explore this possibility, and small-pool PCR procedures to address that task must be validated. An algorithm for the use of small-pool PCR to identify patients with FCCX having such attenuating mutations is put forward as a means of detecting single nucleotide polymorphisms (SNPs) for the future diagnosis of cancer in those at risk.

Determination of Genetically Modified Soybean by Multiplex PCR and CGE with LIF Detection

A method for rapid screening, identification and detection of genetically modified soybean by multiplex polymerase chain reaction (PCR) and capillary gel electrophoresis with laser-induced fluorescence (CGE–LIF) was developed and applied to actual food samples. A triplex PCR procedure was used to amplify the parts of nopaline synthase (NOS) terminator, and the junction between cauliflower mosaic virus 35S promoter and chloroplast transit peptide CTP4 trait gene, as well as the lectin gene to allow the screening and identification of specific transgenic soybean line (glyphosate-tolerant soybean). The multiplex PCR parameters and conditions of capillary gel electrophoresis were optimized. The amplified DNA fragments were analyzed by CGE–LIF. The amplified PCR products were analyzed by CGE–LIF within about 20 min. The method developed is highly sensitive and allows the detection of a percentage of genetically modified soybean as low as 0.025%. The percentage is low enough to fulfill the requirement of the EU Regulation for transgenic food labeling of 1.0%. The sequences of the multiple PCR products were identical with those published in Genbank. The proposed method has been used in identification and detection of genetically modified soybean in various food samples. Compared with agarose gel electrophoresis (AGE), the proposed method is more rapid, accurate and requires a smaller amount of samples. Thus an efficient alternative method is provided for monitoring genetically modified soybean in order to meet the increasing demand of implementation of the genetically modified food labeling policy.

Pneumonia of Turkey Breeder Hens Associated with Mycoplasma Synoviae

Turkey breeder hens showed an increase in mortality beginning at 38 wk of age with no other clinical signs or changes in egg production. While no respiratory signs were observed in live turkeys, those that died consistently had gross lesions of pneumonia. Histopathology of lungs revealed serofibrinous bronchopneumonia, lymphofollicular reaction, and other features suggesting a bacterial etiology. However, except for incidental findings, bacteria were not visualized in the sections examined, and none were isolated in meaningful numbers on routine bacteriologic media. At 42 wk of age the flock showed serologic evidence of infection with Mycoplasma synoviae (MS), and MS was identified by both mycoplasma culture and polymerase chain reaction (PCR) procedures in samples from choanal clefts and tracheas. Results of lung histopathology and PCR tests were consistent with a diagnosis of pneumonia caused by MS.

Preimplantation genetic diagnosis of X-linked adrenoleukodystrophy (ALD) with gender determination using multiple displacement amplification

ALD is an X-linked recessive disorder which is secondary to a mutation in the ABCD1 gene (Xq28) and results neurological effects. The choice method for X-linked ALD-PGD is sexing. But fifty of female embryos are carriers and fifty percent of the discarded male embryos are unaffected diminishing the pool of embryos suitable for transfer. The aim of this work was to increase the reliability of PGD for X-linked ALD and to improve our ability to respond in a fast and safe way thanks to the availability of enough quality DNA obtained by MDA from a single cell for multiple PCR analyses. The MDA product was used for polymerase chain reaction (PCR) analysis of informative markers, used in the PGD–ALD program. A couple which wife carries a causative mutation 676 A > C in the ABCD1 gene that causes X-linked ALD. The affected wife was heterozygous at the DXS1073 and DXS9901 locus. To identify the sex chromosomes X22 informativity test were performed. MDA was used to amplify the whole-genome directly from a single cell. Segregation studies of the family were performed and showed that the ABCD1 extragenic and sex chromosome markers were informative. The development of a MDA–PGD protocol for ALD allowed for the diagnosis of five embryos. The amplification efficiency for DXS1073, DXS9901 and X22 was 12/15, 15/15 and 15/15, respectively. In addition, only ADO in DXS1073 was reported. Four embryos were determined to carry the non-affected haplotype, two embryos were affected, and one could not be diagnosed. Two healthy embryos were transferred 48 hours after culture, resulting in a singleton ongoing pregnancy and the birth of a healthy child. Sex selection for the PGD of X-linked ALD, using standard PCR procedures, has several disadvantages: (1) half of the male embryos could be healthy and will not be replaced, (2) the replacement of carrier female embryos cannot be avoided. This, has led to the fact that, the couples who wish to undergo PGD will rather decide on a specific DNA diagnosis rather than a simple sexing. The availability of enough DNA, thanks to the MDA reaction, allows different target amplification avoiding multiplex optimization protocol, resulting in a very accurate diagnosis. MDA would become a universal first step in PGD. The method we report for the diagnosis of X-linked ALD should be applicable to most patients carrying ALD as long as they were informative for the markers.

Putative virulence genes of Vibrio cholerae from seafoods and the coastal environment of Southwest India

Shrimp, clam and oysters were obtained at two fish markets and at a fish landing dock, and plankton, water and sediment samples were obtained from four river estuaries, in southern India. The samples were analyzed for Vibrio cholerae by conventional isolation techniques and by polymerase chain reaction (PCR) procedures. V. cholerae was isolated from 2 of 5 shrimp, 2 of 5 clam and 5 of 20 water samples. All biochemically confirmed isolates of V. cholerae were positive for toxR. For direct detection of V. cholerae in enrichment broths, PCR was performed using lysates from 0 and 6 h enrichments. All the V. cholerae isolates and enrichment broth lysates were subjected to PCR analysis for the detection of the genes toxR, ctxA, tcpA, ompU, hly, ace, Nag-ST ( stn/sto) , and ompU. Enrichment broths of all the samples which yielded V. cholerae were positive for toxR, OmpU and hlyA genes, while one of a fresh fish market sample was positive for the ace gene. Choleragenic V. cholerae were absent from all environmental samples and fresh fish from the markets, but one sample of shrimp was positive for V. cholerae O139.

Potential microbial risk factors related to soil amendments and irrigation water of potato crops

This study assesses the potential microbial risk factors related to the use of soil amendments and irrigation water on potato crops, cultivated in one traditional and two intensive farms during two harvest seasons. The natural microbiota and potentially pathogenic micro-organisms were evaluated in the soil amendment, irrigation water, soil and produce. Uncomposted amendments and residual and creek water samples showed the highest microbial counts. The microbial load of potatoes harvested in spring was similar among the tested farms despite the diverse microbial levels of Listeria spp. and faecal coliforms in the potential risk sources. However, differences in total coliform load of potato were found between farms cultivated in the autumn. Immunochromatographic rapid tests and the BAM's reference method (Bacteriological Analytical Manual; AOAC International) were used to detect Escherichia coli O157:H7 from the potential risk sources and produce. Confirmation of the positive results by polymerase chain reaction procedures showed that the immunochromatographic assay was not reliable as it led to false-positive results. The potentially pathogenic micro-organisms of soil amendment, irrigation water and soil samples changed with the harvest seasons and the use of different agricultural practices. However, the microbial load of the produce was not always influenced by these risk sources. Improvements in environmental sample preparation are needed to avoid interferences in the use of immunochromatographic rapid tests. The potential microbial risk sources of fresh produce should be regularly controlled using reliable detection methods to guarantee their microbial safety.

Sex-related influence of angiotensin-converting enzyme polymorphisms on fibrosis progression due to recurrent hepatitis C after liver transplantation

Experimental evidence and clinical studies suggest that the renin-angiotensin system and its inhibitors may play a role in regulating the mechanisms of liver fibrosis development. The present study aimed to verify whether carriage of specific angiotensin-converting enzyme (ACE) insertion (I)/deletion (D) allelic variants, modulating angiotensin II generation, could affect the outcome of recurrent hepatitis C after liver transplantation, via several metabolic pathways. Forty-five (29 men) recipients, with a median histological follow-up of 60 months after orthotopic liver transplantation (OLT), were studied. ACE gene I/D polymorphism was assessed by means of a polymerase chain reaction procedure. Fibrosis progression was evaluated annually during the follow-up. Weight gain 1 year post-OLT (defined as an increase in body mass index, BMI, of >0.5 kg/m(2)) was significantly more common among D/ carriers (22/22 vs. 16/23, P < 0.005); patients who 1 year after OLT had an increase in their BMI value of >0.5 kg/m(2) more frequently had a triglycerides/cholesterol ratio of <or= 0.7 (16/22 vs. 8/23, chi-squared test P < 0.02). This association was stronger in men. Female D/D homozygotes had the highest probability of showing significant liver fibrosis (7/10) in comparison with men (11/29) and I/ women (1/6) (P < 0.01). In patients with recurrent hepatitis C, carriers of the D allele appeared to gain more weight after liver transplantation, and in male liver recipients, the D allele was associated with a peculiar lipid profile that was associated with a slower rate of allograft fibrosis progression. Among female recipients, carriage of the D allele may favor more severe allograft fibrosis.

Improved Real-Time Multiplex Polymerase Chain Reaction Detection of Methylenetetrahydrofolate Reductase ( MTHFR) 677C>T and 1298A>C Polymorphisms Using Nearest Neighbor Model-Based Probe Design

The disorders of folate metabolism caused by methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms may lead to several disease states including coronary heart disease, venous thrombosis, and several types of cancer. We have developed a real-time multiplex single-tube polymerase chain reaction procedure on the LightCycler for the detection of the two most commonly occurring variants, 677C>T and 1298A>C, in the MTHFR gene. An improved probe design, based on the nearest neighbor model for nucleic acid-probe duplex stability, resulted in a better separation (DeltaTm approximately 10 degrees C) of melting peaks of the wild-type and mutant alleles than that by the existing method (DeltaTm approximately 3 degrees C) for specimens heterozygous for the 1298A>C polymorphism. Of the 333 blood specimens analyzed by this procedure, we did not find any samples that gave ambiguous results. The specimens with homozygous mutation for one polymorphism were of the wild type for the other variant. The assay was validated by the comparison of the genotyping results of 50 blood specimens from the LightCycler polymerase chain reaction with the conventional restriction fragment length polymorphism procedures. There was 100% concordance of the test results obtained by the two techniques. This assay is reliable, economical, and can be performed by less trained technologists compared with the procedure performed by the conventional restriction fragment length polymorphism technique.

Multicolor real‐time polymerase chain reaction genotyping of six human platelet antigens using displacing probes

Several genotyping methods for six clinically relevant human platelet antigens (HPAs) have been reported. A four-color real-time polymerase chain reaction (PCR) method using displacing probes for genotyping of the six HPAs is described. Primers and four differently fluorophor-labeled displacing probes were designed and synthesized to detect single-nucleotide polymorphisms responsible for each of the HPA-1, -2, -3, -4, -5, and -15 genotypes. Two HPA systems were analyzed in a single PCR procedure. After validation with samples of known genotypes, a total of 150 blood samples from healthy donors were genotyped. The results were compared with PCR with sequence-specific primers (SSP), PCR-restriction fragment length polymorphism (RFLP), and/or direct DNA sequencing. The frequencies of each HPA allele were calculated. Unequivocal real-time PCR genotyping results were obtained with minimal manual manipulation and carryover contamination. All 150 blood samples were correctly genotyped as confirmed by PCR-SSP, PCR-RFLP, and/or direct DNA sequencing. The allelic frequencies of HPA-1 through -5 and -15 among the Chinese population in Xiamen were comparable with those previously reported with Chinese living in other territories. For each specimen, genotyping of all six HPA biallelic systems was achieved in three tubes of PCR within 90 minutes and with material cost of no more than $1. Genotyping of HPA with real-time PCR using displacing probes is more rapid and reliable compared with PCR-SSP and PCR-RFLP methods and is more affordable than existing real-time PCR-based HPA genotyping assays. Thus, our approach is more suitable for routine HPA analysis and ideal for both urgent clinical testing and high-throughput screening.

Amplification of c‐myc and cyclin D1 genes in primary and metastatic carcinomas of the liver

The c-myc and cyclin D1 genes are included among the oncogenes the amplifications of which have been detected in cancers of various organs. However, there have been few reports on the amplification of both these genes in primary and metastatic liver carcinomas. In the present study, c-myc and cyclin D1 gene amplification was examined in 76 primary and metastatic liver carcinomas using formalin-fixed paraffin-embedded tissue sections and a differential polymerase chain reaction procedure. c-myc and cyclin D1 gene amplification was detected in 15 (33%) and two (4%) of 46 hepatocellular carcinomas (HCC), one (10%) and 0 (0%) of 10 intrahepatic cholangiocarcinomas (ICC), one (33%) and 0 (0%) of three combined hepatocellular and cholangiocarcinomas (HCC + ICC), and nine (56%) and three (19%) of 16 metastatic lesions to the liver from colorectal adenocarcinoma (MCA), respectively. The incidence of c-myc amplification was significantly higher in MCA than in ICC (P = 0.023), and it tended to be higher in HCC than in ICC. These results indicate that the amplification of the c-myc proto-oncogene is not unusual in HCC and MCA, and its detection may have a useful diagnostic significance in differentiating ICC from MCA or HCC from ICC.

PCR‐based markers for detection of different sources of 1AL.1RS and 1BL.1RS wheat–rye translocations in wheat background

AbstractThe rye (Secale cereale L.) chromosome arm 1RS is one of the most successfully used alien resources in wheat (Triticum aestivum L.) improvement, and it is still being widely utilized by many breeding programmes. With increasing application of marker‐assisted selection in wheat breeding, development of an efficient molecular marker system to monitor and track 1AL.1RS and 1BL.1RS wheat–rye translocations is of practical value. In this study, we systematically evaluated the utility of eight rye‐specific molecular markers in detecting 1RS chromatins with different origins in diverse wheat genetic backgrounds. Two such markers, PAWS5/S6 and SCM9 were identified that were able to differentiate multiple sources of wheat–rye translocations involving 1RS. A duplex polymerase chain reaction (PCR) procedure was developed with two rye‐specific markers PAWS5/S6 and RIS and tested in a set of representative wheat lines. The two rye‐specific markers and the duplex PCR procedure established in this study provided a useful tool in marker‐assisted selection of materials containing desirable 1RS chromatin in wheat breeding.

Detection of Phytophthora melonis in samples of soil, water, and plant tissue with polymerase chain reaction

A polymerase chain reaction (PCR) assay for the detection of Phytophthora melonis, the causal agent of phytophthora blight of cucumber, was developed. PCR primers specific to P. melonis (Pm1 and Pm2) were designed using sequences of the internal transcribed spacer (ITS) of nuclear ribosomal DNA. More than 115 isolates representing 26 species of Phytophthora and 29 other species of pathogens were used to test the specificity of the primers. PCR amplification with these primers resulted in a product of ca 545 base pairs exclusively for isolates of P. melonis. The detection sensitivity with P. melonis primers was 100 fg of genomic DNA. A nested PCR procedure with DC6 and ITS4 as first-round primers, followed by Pm1 and Pm2 primers, increased detection sensitivity 1000-fold to 100 ag. With nested PCR, the detection sensitivity for the soil pathogens was 10 zoospores in 0.5 g of artificially inoculated soil. PCR with Pm1 and Pm2 was also used to detect P. melonis in naturally infected cucumber tissue, soil, and irrigation water. Real-time fluorescent quantitative PCR assays were developed to detect and monitor the pathogen in plant samples. The PCR-based methods developed could simplify both plant disease diagnosis and pathogen monitoring as well as help guide plant disease management.

Prevalence of Cholesteryl Ester Storage Disease

Cholesteryl ester storage disease (CESD) is an autosomal recessive chronic liver disease caused by lysosomal acid lipase (LAL) deficiency. The gene is located on chromosome 10q23.2-q23.3, and the enzyme is essential for triglycerides and cholesteryl ester hydrolysis in lysosomes. CESD is characterized by hypercholesterolemia, hypertriglyceridemia, HDL deficiency, and abnormal lipid deposition in many organs. In the liver this results in hepatomegaly caused by hepatic steatosis and fibrosis that can lead to micronodular cirrhosis.1 Disease onset takes place during childhood or adolescence. Males and females are affected in about equal numbers. Patients rarely reach the age of 30. Biochemically, the disorder is recognized by largely reduced lysosomal acid lipase activity.2,3 Complete absence of LAL activity causes Wolman Disease, which is normally fatal within the first 6 months of life.1,4 Several groups have identified mutations in the LAL gene underlying CESD and Wolman disease.5–9 Mutations causing Wolman disease produce an enzyme with no residual activity or no enzyme at all, whereas CESD-causing mutations encode for LAL which retains some enzyme activity.4,10 A G-to-A transition at position −1 of the exon 8 splice donor (E8SJM, E xon 8 S plice J unction M utation) leads to an in-frame deletion of exon 8. The resulting protein is 24 amino acids shorter and has no residual LAL activity, however E8SJM does not cause Wolman Disease because 2% to 4% of normally spliced LAL is present in homozygote carriers.11,12 The vast majority of CESD …

An evaluation of primers amplifying DNA targets for the detection of Cryptosporidium spp. using C. parvum HNJ-1 Japanese isolate in water samples

The performance of polymerase chain reaction (PCR) procedures for the detection of Cryptosporidium parvum HNJ-1 strain (genotype II) oocysts purified from mice using published protocols was evaluated. Oocysts were concentrated from fecal samples of infected severe combined immunodeficiency (SCID) mice by sucrose flotation and were then purified by immunomagnetic separation method. The genotype of C. parvum was established as type II by restriction fragment length polymorphism (RFLP) analysis. Water samples were spiked with different numbers of oocysts, determined by limiting dilution. Genomic DNA was extracted and used for PCR assays targeting various Cryptosporidium species genes (Beta-Tubulin, COWP, 70 kDa HSP, SSU rRNA, ITS1, TRAP-C1 and TRAP-C2 gene). DNA from oocyst numbers of more than 1 x 10(4) was detected using each of the primers. However, when using lower oocyst numbers, the tools based on 9 of the 16 different primer assays gave sufficient results. Assays using the remaining seven primers gave less than satisfactory results. A new primer set, named VKSS-F1/2 and VKSS-R1/2, that target the 18 SSU rRNA gene of C. parvum was constructed and applied. The VKSS-F1/2 and VKSS-R1/2 assays amplified DNA isolated from spiked samples in 206 of 211 trials (97.6%). This illustrates the difficulty of detecting low numbers of Cryptosporidium spp. oocysts by molecular methods when working with environmental samples.

Differential signalling pathways involved in cholinoceptor‐dependent stimulation of nitric oxide isoforms in dental pulp

To investigate the role of muscarinic acetylcholine receptor (mAChR) subtype activity in the regulation of endothelial- (e) and neuronal- (n) nitric oxide synthase (NOS) expression and activity. Rat dental pulp tissue was used throughout the study. The e-nos and n-nos mRNA levels were specifically measured using reverse transcriptase polymerase chain reaction procedures that involve simultaneous co-amplification of both target cDNA and a reference template with a single set of primers. NOS activity was measured by the production of [U-(14)C]-citrulline from [U-(14)C]-arginine. Stimulation of M(1)/M(2) and M(3)/M(4) mAChRs with pilocarpine caused an increase in e-nos and n-nos mRNA levels and NOS activity in the dental pulp. The specific mAChR subtype antagonists, L-NMMA, l-NIO and N(2)-propyl-L-arginine but not aminoguanidine attenuated all these effects. Inhibitors of phospholipase C (PLC), protein kinase C (PKC) and calcium/calmodulin (CaM) prevented the pilocarpine-dependent increase in n-nos and e-nos mRNA levels and NOS activity. Activation of mAChR subtypes stimulated NOS activity by increasing the production of NO through e-nos and n-nos gene expression and NOS activity. The mechanism appears to occur secondarily to stimulation of CaM and PKC enzymatic activity.