Molecular testing for synovial sarcoma.
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Comment↗
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Results for “molecular testing”
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Development of molecular tests for the detection of ILAR and latent viruses in fruit trees.
The detection throughout the year of latent and ILAR viruses in fruit tress by classical serological tests appear to be unreliable. We have developed RT-PCR tests for a reliable detection of latent and ILAR viruses in fruit trees. These assays were then simplified to allow the direct use of crude plant extracts instead of total RNA preparations, and the analyses of pooled samples. In this way, such RT-PCR protocols are suitable for a routine diagnosis of latent and ILAR viruses in fruit tree certification.
Fruit↗
[Biological diagnosis of hepatitis C virus infection. Role of serological and molecular tests].
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Enzyme-Linked Immunosorbent Assay↗
[A molecular test for the detection of E. coli F18 receptors: a breakthrough in the struggle against edema disease and post-weaning diarrhea in swine].
Oedema disease and post-weaning diarrhoea in swine are associated with the colonization of the intestine with toxigenic Escherichia (E.) coli bacteria of various serotypes. Colonization depends on specific binding between adhesive fimbriae and receptors on the enterocytes. The demonstration of these receptors allows the identification of susceptible and resistant pigs. Direct sequencing of the alpha (1,2) fucosyltransferase gene (FUT1) in swine being either susceptible or resistant to adhesion by F18 fimbriated E.coli revealed a mutation at basepair 307 (M307). Analysis of the mutation in Swiss Landrace and Large White families showed close linkage with the locus controlling resistance and susceptibility to E.coli F18 adhesion (ECF18R). The FUT1 (M307) mutation is a good marker for selection of E.coli of F18 adhesion resistant animals. The mutation is found with variable frequencies in Duroc, Hampshire and Pietrain pigs as well.
Animals↗
Optimized procedure for DNA isolation from fresh and cryopreserved clotted human blood useful in clinical molecular testing.
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Blood Coagulation↗
Ethical issues in DNA testing.
Molecular tests for single gene disorders have become the "gold standard," surpassing in sensitivity and specificity other ancillary tests such as imaging or neurophysiologic tests. Although direct technical problems are rare for DNA tests, few outcomes studies have been reported that examine sensitivity, specificity, and usage of tests outside the setting of academic medical centers. Ethical problems of DNA testing are mainly discussed in a theoretical framework and few published studies or case reports deal with real-life situations. Among neurologists, ethical dilemmas and attempts at their resolution are communicated mainly on a verbal basis, and even then relatively little follow-up is available. The potential use of information obtained through DNA testing by other family members, employers, insurance companies, and governmental agencies deserves scientific investigation and societal discussion. A major educational effort is needed to inform patients, providers, and third parties that the presence of a particular allele in an individual is only one piece in a complex puzzle of environmental and genetic interactions that may ultimately result in disease.
DNA↗
Principles of molecular microbiology testing methods.
Molecular testing methods have the potential to replace many conventional microbiology laboratory assays. Recent refinements in technology have resulted in more user-friendly testing platforms. These platforms are automated and have lowered risks for contamination, decreased costs, and are faster than older platforms. The success of these technologies depends on their successful application to patient care. Quality issues include appropriate specimens for analysis, performance characteristics of different analytical methods, optimal specimen processing, the effects of PCR inhibitors, and false-positive results caused by contaminating nucleic acids. Quality control guidelines for molecular microbiologic diagnostic assays are in their infancy and require further development. Additionally, the problem of "too much" sensitivity (brought on by the extreme sensitivity of these techniques coupled with the potential presence of small numbers of pathogenic organisms in asymptomatic individuals) should be considered. Potential problems when monitoring therapy (because molecular detection techniques do not generally have the ability to determine whether an organism is dead or alive) can also occur. Cost-effective test use, pathogen- or disease-targeted algorithms, and standardized methods will be necessary for the true value of these technologies to be realized. This is especially important, because, unlike traditional culture methods, most molecular microbiology methods are pathogen-specific. Clinicians familiar with the reasons why "pan-culture" (i.e., requesting all culture possibilities at once) is inadvisable should not use the same irrational approach when requesting molecular tests. The clinical usefulness of molecular testing will be maximized as targeted algorithms are developed and an understanding of molecular test ordering patterns is realized. Laboratory technicians and physicians must continue to apply and combine theories of traditional microbiology, clinical chemistry, and general medicine to the understanding and application of molecular diagnostics.
Base Sequence↗
Is the DNA sequence the gold standard in genetic testing? Quality of molecular genetic tests assessed.
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Benchmarking↗
Relative accuracy of the halothane challenge test and a molecular genetic test in detecting the gene for porcine stress syndrome.
Pietrain swine homozygous for the hal gene (n) associated with porcine stress syndrome (PSS) and a Pietrain-derivative breed, Near Pietrain (NP), with a frequency of .31 for n, were mated to produce reciprocal F1, F2, and purebred NP litters. The halothane challenge test was performed on all 40 parents and 240 progeny to predict their hal genotype and PSS susceptibility. The DNA-based assay for a C to T mutation at base pair 1,843 of the skeletal muscle ryanodine receptor (ryr1) cDNA, which is very highly correlated with PSS, was also determined for these animals. The predicted hal genotypes observed in the progeny differed significantly from the expected Mendelian ratios, and the halothane challenge test consistently overestimated the n/n hal genotype. However, the ryr1 genotypes observed in the progeny did not differ significantly from the expected Mendelian ratios, and this DNA-based assay apparently misidentified only one of the 40 parents. The results of this study indicate that the assay for the ryr1 mutation more accurately predicts both the homozygous and heterozygous forms of the PSS gene than does the halothane challenge test.
Animals↗
Human papillomaviruses (HPV) in gynaecological cytology: from molecular biology to clinical testing.
Molecular epidemiological and pathological studies show that different HPV types are associated with different cervical lesions allowing classification of the viruses into types associated with 'high', 'intermediate' and 'low' risk of cervical neoplasia. However, HPV infection often regresses and, where it is associated with neoplasia, is an early event. This suggests that other factors are involved in the carcinogenic process, and there is some mechanistic basis for the interaction of epidemiologically defined factors with HPV infection in the process of cervical carcinogenesis. With the refinement of techniques for HPV detection in clinical material, HPV testing is now a realistic possibility, but how this should be performed and in what clinical situation(s) is still uncertain. Particular areas of interest are: (i) the assessment of patients with borderline cytological changes or mild dyskaryosis; and (ii) the definition of those patients at greater risk of invasive disease. Clinical trials are needed before the utility of HPV testing can be properly assessed.
Cell Transformation, Neoplastic↗
Diagnosis of HIV infection and laboratory monitoring of its therapy.
BACKGROUND: Serological diagnosis of human immunodeficiency virus (HIV) infection became available in 1985, with the rapid increase in sensitivity and specificity of enzyme-linked immunosorbent assays (ELISAs) and the supplement tests. Molecular tests for detection of HIV in the diagnosis of HIV infection in special settings and monitoring of HIV-1 infection followed this. OBJECTIVE AND DESIGN: In this review it is intended to give a brief overview of the diagnosis and monitoring of HIV infection. RESULTS AND CONCLUSION: Serological methods and molecular methods for the detection and quantitation of HIV are discussed.
Clinical Laboratory Techniques↗
Family and population strategies for screening and counselling of inherited cardiac arrhythmias.
Family screening in inherited cardiac arrhythmias has been performed in The Netherlands since 1996, when diagnostic DNA testing in long QT syndrome (LQTS) and hypertrophic cardiomyopathy (HCM) became technically possible. In multidisciplinary outpatient academic clinics, an adjusted protocol for genetic counselling, originally derived from predictive testing in Huntington's disease, is being used. 1110 individuals, including 842 relatives of index patients, were informed about their risks, and most were tested molecularly and/or clinically for carriership of the disease present in their family. Of 345 relatives who were referred for cardiologic follow-up, 189 are being treated, because of an increased risk of life-threatening arrhythmias. Evaluation of the psychological and social consequences of family screening for inherited arrhythmias can be performed by using the adapted criteria of Wilson and Jüngner, i.e., from a point of view of public health. Preliminary results of psychological research show that parents of children at risk for LQTS show high levels of distress. Many other aspects have to be evaluated yet, making final conclusions about the feasibility of family screening difficult, particularly in HCM. Clinical guidelines are urgently needed. Population screening by molecular testing, for instance in athletic preparticipation screening, will become possible in the future and has its own prerequisites for success.
Arrhythmias, Cardiac↗
[Molecular diagnostic tests and chromosome analyses].
Molecular diagnostic tests and chromosomal analyses are often used to diagnose infectious, neoplastic and genetic diseases. However, there are only a few genetic laboratories in university hospitals in Japan because of the high cost and additional staff required for their management. In 1992, we started a genetic laboratory with one technologist in Shinshu University Hospital, and mainly performed polymerase chain reactions(PCR) for the diagnosis of several mycobacterial species, Hepatitis C virus and cytomegalovirus. Infectious diseases diagnosed by PCR have gradually increased over recent years. Chromosome analyses were available in 1996 and spectral karyotyping started from April 1999. Our genetic laboratory was staffed by 4 technologists and 3 faculty members in 1999, and they can provide informative tests with high quality. Molecular diagnostic tests were performed for 855 infectious diseases, 25 hematopoietic tumors and 8 solid tumors, while chromosome analyses were performed for 51 hereditary diseases and 29 hematopoietic tumors in 1998. Establishment of a genetic laboratory in a university hospital may not result in cost savings, but it produces a favorable influence on other laboratories. All staff can use molecular biology techniques to clarify limitations of routine tests and research.
Chromosomes↗
The UK National External Quality Assessment Scheme (UK NEQAS) for molecular genetic testing in haemophilia.
Molecular genetic analysis of families with haemophilia and other inherited bleeding disorders is now a common laboratory investigation. In contrast to phenotypic testing in which strict quality control is adhered to, in haemophilia molecular genetic testing there has been a lack of any external quality assurance schemes. In 1998 the UK National External Quality Assessment Scheme (UK NEQAS) established a pilot quality assurance scheme for molecular genetic testing in haemophilia. Results from three initial surveys highlighted problems with the quality of samples when used to screen for the intron 22 inversion within the F8 gene. The scheme was re-launched in 2003, and since that time there have been five exercises involving whole blood or immortalised cell line DNA. The results together with an overall summary of the exercise are subsequently returned to participants. Exercises to date have focused exclusively on haemophilia A and QA, material has included screening for the intron 1 and intron 22 inversions as well as sequence analysis. A paper exercise circulated in 2003 highlighted problems with the format of reports and, following feedback to participants, only a single error has been made in the subsequent four exercises. Participating laboratories now receive QA material every six months. Immortalised cell line material was introduced in 2005 and was shown to perform well. This will allow expansion of the scheme and a reduction in the dependence on blood donation.
Chromosome Inversion↗
Detection of an MEN1 gene mutation depends on clinical features and supports current referral criteria for diagnostic molecular genetic testing.
OBJECTIVE: Diagnostic molecular genetic testing for multiple endocrine neoplasia type 1 (MEN1) has been available since the identification of the MEN1 gene in 1997. Mutation screening of the MEN1 gene has been recommended for patients who meet clinical criteria for MEN1 (at least two of the following: parathyroid hyperplasia, pancreatic endocrine tumour or pituitary adenoma) and those in whom a diagnosis of MEN1 is suspected. We examined the appropriateness of these clinical criteria. PATIENTS AND METHODS: A total of 292 patients were referred for diagnostic testing. The coding region of the MEN1 gene was sequenced in 186 index cases and mutation testing was requested for 106 subjects, including 83 asymptomatic relatives. RESULTS: MEN1 gene mutations were identified in 68/186 index cases (37%). Twenty-nine of the 60 MEN1 mutations reported are novel. The likelihood of finding a mutation was correlated with the number of MEN1-related tumours (mutation detection rate of 79%, 37% and 15% in patients with three, two and one main MEN1-related tumours; P < or = 0.00001) and increased in the presence of a family history (mutation detection rate of 91%, 69% and 29%vs. 69%, 23% and 0% in sporadic cases with three, two or one main MEN1-related tumours, respectively; P < or = 0.00001). The pick-up rate in the 83% of subjects who met proposed criteria for diagnostic testing was 42%, but in those who did not meet these criteria this fell to 0%. CONCLUSIONS: The likelihood of finding an MEN1 mutation depends on the clinical features of the patient and their family. This large series supports present referral criteria for diagnostic mutation screening, but suggests that patients with sporadic isolated tumours rarely have MEN1 mutations.
Adolescent↗
Comparison of functional testing for resistance to activated protein C and molecular biological testing for factor V R506Q in 370 patients.
OBJECTIVE: To compare functional and molecular biological tests for resistance to activated protein C (APC)/factor V R506Q, the most common cause of familial thrombosis. METHODS: We developed functional and molecular biological tests for resistance to APC/factor V R506Q at our institution and correlated the results for 370 patients studied by both methods. The functional method is based on addition of exogenous APC to an activated partial thromboplastin time-based assay. The molecular biological method is based on polymerase chain reaction followed by endonuclease digestion. RESULTS: Considering the molecular biological test as definitive for detecting the factor V R506Q mutation, the sensitivity of the functional assay was 100%, and the specificity was 74%. The prevalence of the factor V mutation in the population studied was 12% (41 heterozygotes, two homozygotes), and the positive predictive value of the functional assay was 34%. Although a normalized sensitivity ratio (nAPC-SR) less than 0.84 is considered evidence of resistance to APC by functional testing, we found that all patients with factor V R506Q had an nAPC-SR less than or equal to 0.71. When this alternative positive cutoff was used, the specificity of the functional test for factor V R506Q increased to 87%, and the positive predictive value increased to 52%, which constituted a significant improvement. We compared clinical findings from patients with resistance to APC with or without the presence of factor V R506Q, and found that as a group, those with factor V R506Q had a higher incidence of hypercoagulability, but fewer additional risk factors for hypercoagulability. The mechanism of resistance to APC in factor V R506Q-negative individuals is unclear, but may be related to other risk factors for hypercoagulability. CONCLUSIONS: The functional assay for resistance to APC is an excellent screening test for factor V R506Q, but confirmatory molecular biological testing is necessary when the functional test is positive, because of the high false-positive rate.
Adult↗
Design, validation, and absolute sensitivity of a novel test for the molecular detection of avian pneumovirus.
This study describes attempts to increase and measure sensitivity of molecular tests to detect avian pneumovirus (APV). Polymerase chain reaction (PCR) diagnostic tests were designed for the detection of nucleic acid from an A-type APV genome. The objective was selection of PCR oligonucleotide combinations, which would provide the greatest test sensitivity and thereby enable optimal detection when used for later testing of field materials. Relative and absolute test sensitivities could be determined because of laboratory access to known quantities of purified full-length DNA copies of APV genome derived from the same A-type virus. Four new nested PCR tests were designed in the fusion (F) protein (2 tests), small hydrophobic (SH) protein (1 test), and nucleocapsid (N) protein (1 test) genes and compared with an established test in the attachment (G) protein gene. Known amounts of full-length APV genome were serially diluted 10-fold, and these dilutions were used as templates for the different tests. Sensitivities were found to differ between the tests, the most sensitive being the established G test, which proved able to detect 6,000 copies of the G gene. The G test contained predominantly pyrimidine residues at its 3' termini, and because of this, oligonucleotides for the most sensitive F test were modified to incorporate the same residue types at their 3' termini. This was found to increase sensitivity, so that after full 3' pyrimidine substitutions, the F test became able to detect 600 copies of the F gene.
Animals↗
Achieving quality reproducible results and maintaining compliance in molecular diagnostic testing of human papillomavirus.
Laboratories contemplating either the addition of new molecular tests or modifying methods approved by the Food and Drug Administration for human papillomavirus testing should be aware of a variety of procedural, performance, and regulatory issues surrounding such activity. Diagnostic medical laboratory testing in the United States is regulated by the Centers for Medicare and Medicaid Services, an agency formerly known as the Health Care Finance Administration. The regulatory vehicle of the Centers for Medicare and Medicaid Services is manifested in the Clinical Laboratory Improvement Amendments (CLIA). The CLIA program has put into place specific regulations for laboratory quality control, which includes specific recommendations for method validation. Regulations that must be followed regarding personnel, quality control, quality assurance, method validation, and proficiency testing depend on the complexity category of the individual test. All molecular diagnostic tests, including those for human papillomavirus, are considered high complexity. The Centers for Medicare and Medicaid Services retains the authority to allow private, national accreditation organizations to "deem" that a laboratory is compliant with CLIA '88 requirements. Accreditation organizations, such as the Joint Commission for Accreditation of Hospitals, the Commission on Office Laboratory Accreditation, and the College of American Pathologists (CAP), as well as several state medical laboratory-accrediting agencies, possess the authority to deem laboratories as "CLIA-approved." The CAP, through its Laboratory Accreditation Program, has promoted standards for laboratory performance and method validation. In general, guidelines set forth in the CAP Laboratory Accreditation Program checklists specify that all clinical laboratory testing must essentially meet those requirements defined for high-complexity testing under CLIA '88, including test validation standards, reportable/reference ranges, performance criteria, and proficiency testing.
Female↗