Variation in pre-PCR processing of FFPE samples leads to discrepancies in BRAF and EGFR mutation detection: a diagnostic RING trial.

AIMS: Mutation detection accuracy has been described extensively; however, it is surprising that pre-PCR processing of formalin-fixed paraffin-embedded (FFPE) samples has not been systematically assessed in clinical context. We designed a RING trial to (i) investigate pre-PCR variability, (ii) correlate pre-PCR variation with EGFR/BRAF mutation testing accuracy and (iii) investigate causes for observed variation. METHODS: 13 molecular pathology laboratories were recruited. 104 blinded FFPE curls including engineered FFPE curls, cell-negative FFPE curls and control FFPE tissue samples were distributed to participants for pre-PCR processing and mutation detection. Follow-up analysis was performed to assess sample purity, DNA integrity and DNA quantitation. RESULTS: Rate of mutation detection failure was 11.9%. Of these failures, 80% were attributed to pre-PCR error. Significant differences in DNA yields across all samples were seen using analysis of variance (p

Application of self-quenched JH consensus primers for real-time quantitative PCR of IGH gene to minimal residual disease evaluation in multiple myeloma.

Monitoring multiple myeloma patients for relapse requires sensitive methods to measure minimal residual disease and to establish a more precise prognosis. The present study aimed to standardize a real-time quantitative polymerase chain reaction (PCR) test for the IgH gene with a JH consensus self-quenched fluorescence reverse primer and a VDJH or DJH allele-specific sense primer (self-quenched PCR). This method was compared with allele-specific real-time quantitative PCR test for the IgH gene using a TaqMan probe and a JH consensus primer (TaqMan PCR). We studied nine multiple myeloma patients from the Spanish group treated with the MM2000 therapeutic protocol. Self-quenched PCR demonstrated sensitivity of >or=10(-4) or 16 genomes in most cases, efficiency was 1.71 to 2.14, and intra-assay and interassay reproducibilities were 1.18 and 0.75%, respectively. Sensitivity, efficiency, and residual disease detection were similar with both PCR methods. TaqMan PCR failed in one case because of a mutation in the JH primer binding site, and self-quenched PCR worked well in this case. In conclusion, self-quenched PCR is a sensitive and reproducible method for quantifying residual disease in multiple myeloma patients; it yields similar results to TaqMan PCR and may be more effective than the latter when somatic mutations are present in the JH intronic primer binding site.

Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936.

In a European BIOMED-2 collaborative study, multiplex PCR assays have successfully been developed and standardized for the detection of clonally rearranged immunoglobulin (Ig) and T-cell receptor (TCR) genes and the chromosome aberrations t(11;14) and t(14;18). This has resulted in 107 different primers in only 18 multiplex PCR tubes: three VH-JH, two DH-JH, two Ig kappa (IGK), one Ig lambda (IGL), three TCR beta (TCRB), two TCR gamma (TCRG), one TCR delta (TCRD), three BCL1-Ig heavy chain (IGH), and one BCL2-IGH. The PCR products of Ig/TCR genes can be analyzed for clonality assessment by heteroduplex analysis or GeneScanning. The detection rate of clonal rearrangements using the BIOMED-2 primer sets is unprecedentedly high. This is mainly based on the complementarity of the various BIOMED-2 tubes. In particular, combined application of IGH (VH-JH and DH-JH) and IGK tubes can detect virtually all clonal B-cell proliferations, even in B-cell malignancies with high levels of somatic mutations. The contribution of IGL gene rearrangements seems limited. Combined usage of the TCRB and TCRG tubes detects virtually all clonal T-cell populations, whereas the TCRD tube has added value in case of TCRgammadelta(+) T-cell proliferations. The BIOMED-2 multiplex tubes can now be used for diagnostic clonality studies as well as for the identification of PCR targets suitable for the detection of minimal residual disease.

Incomplete DJH rearrangements as a novel tumor target for minimal residual disease quantitation in multiple myeloma using real-time PCR.

The hypervariable regions of immunoglobulin heavy-chain (IgH) rearrangements provide a specific tumor marker in multiple myeloma (MM). Recently, real-time PCR assays have been developed in order to quantify the number of tumor cells after treatment. However, these strategies are hampered by the presence of somatic hypermutation (SH) in VDJH rearrangements from multiple myeloma (MM) patients, which causes mismatches between primers and/or probes and the target, leading to a nonaccurate quantification of tumor cells. Our group has recently described a 60% incidence of incomplete DJH rearrangements in MM patients, with no or very low rates of SH. In this study, we compare the efficiency of a real-time PCR approach for the analysis of both complete and incomplete IgH rearrangements in eight MM patients using only three JH consensus probes. We were able to design an allele-specific oligonucleotide for both the complete and incomplete rearrangement in all patients. DJH rearrangements fulfilled the criteria of effectiveness for real-time PCR in all samples (ie no unspecific amplification, detection of less than 10 tumor cells within 10(5) polyclonal background and correlation coefficients of standard curves higher than 0.98). By contrast, only three out of eight VDJH rearrangements fulfilled these criteria. Further analyses showed that the remaining five VDJH rearrangements carried three or more somatic mutations in the probe and primer sites, leading to a dramatic decrease in the melting temperature. These results support the use of incomplete DJH rearrangements instead of complete somatically mutated VDJH rearrangements for investigation of minimal residual disease in multiple myeloma.

Heteroduplex PCR analysis of rearranged immunoglobulin genes for clonality assessment in multiple myeloma.

BACKGROUND AND OBJECTIVE: Molecular analysis by PCR of monoclonally rearranged immunoglobulin (Ig) genes can be used for diagnosis in B-cell lymphoproliferative disorders (LPD), as well as for monitoring minimal residual disease (MRD) after treatment. This technique has the risk of false-positive results due to the "background" amplification of similar rearrangements derived from polyclonal B-cells. This problem can be resolved in advance by additional analyses that discern between polyclonal and monoclonal PCR products, such as the heteroduplex analysis. A second problem is that PCR frequently fails to amplify the junction regions, mainly due to somatic mutations frequently present in mature (post-follicular) B-cell lymphoproliferations. The use of additional targets (e.g. Ig light chain genes) can avoid this problem. DESIGN AND METHODS: We studied the specificity of heteroduplex PCR analysis of several Ig junction regions to detect monoclonal products in samples from 84 MM patients and 24 patients with B cell polyclonal disorders. RESULTS: Using two distinct VH consensus primers (FR3 and FR2) in combination with one JH primer, 79% of the MM displayed monoclonal products. The percentage of positive cases was increased by amplification of the Vlamda-Jlamda junction regions or kappa(de) rearrangements, using two or five pairs of consensus primers, respectively. After including these targets in the heteroduplex PCR analysis, 93% of MM cases displayed monoclonal products. None of the polyclonal samples analyzed resulted in monoclonal products. Dilution experiments showed that monoclonal rearrangements could be detected with a sensitivity of at least 10(-2) in a background with >30% polyclonal B-cells, the sensitivity increasing up to 10(-3) when the polyclonal background was

Anti-N-methyl-D-Aspartate Receptor Encephalitis with Positive Serum Antithyroid Antibodies, IgM Antibodies Against Mycoplasma Pneumoniae and Human Herpesvirus 7 PCR in the CSF

We report the case of a boy with an encephalopathy associated with extrapyramidal and psychiatric symptoms and anti-N-methyl-D-aspartate receptor antibodies. He had positive serum antithyroid antibodies, IgM antibodies against Mycoplasma pneumoniae and human herpesvirus 7 polymerase chain reaction in the cerebrospinal fluid. He was successfully treated with rituximab, after steroids, intravenous immunoglobulin and plasma exchange. The pathophysiology of this disorder may be post-infectious and autoimmune.

Study of infectious haematopoietic necrosis disease in some hatcheries and farmed rainbow trout in Iran using immunohistochemical technique and to comparing the obtained results with the PCR works

To detect rainbow trout hatcheries for infectious hematopoietic necrosis virus, samples of kidney, liver, spleen, muscle, intestine, heart and gills of trout larvae were obtained from a number of trout hatcheries from different provinces. Also tissue samples were obtained for molecular works using RT- PCR procedure. Tissue samples were processed using standard histotechnique and the obtained sections were stained using immunohistochemical procedure. From 100 examined samples 35 were positive for IHN by immunohistochemical test. Also, from 100 samples examined, 43 were positive in RT- PCR studies. The obtained results show that some rainbow trout hatcheries are contaminated in different regions of country. Therefore, a definition of prevention and eradication criteria are now critical to protect the unaffected areas within the country.

Study of white spot disease in four native species in Persian Gulf by histopathology and PCR methods

After serious disease outbreak, caused by new virus (WSV), has been occurring among cultured penaeid shrimps in Asian countries like China since 1993 and then in Latin American countries, during June till July 2002 a rapid and high mortality in cultured Penaeus indicus in Abadan region located in south of Iran with typical signs and symptoms of White Spot Syndrome Virus was confirmed by different studies of Histopathology, PCR, TEM, Virology. This study was conducted for the purpose of determination of prevalence(rate of infection)/ROI and grading severity (SOI) of WSD to five species: 150 samples of captured shrimps and 90 samples of cultured ones; Penaeus indicus, P. semisulcatus, P. merguiensis, Parapenaopsis styliferus, and Metapenaeus affinis in 2005. 136 of 240 samples have shown clinical and macroscopical signs & symptoms including; white spots on carapase (0.5-2 mm), easily removing of cuticule, fragility of hepatopancreas and red color of motility limbs. Histopathological changes like specific intranuclear inclusion bodies (cowdry-type A) were observed in all target tissues (gill, epidermis, haemolymph and midgut) but not in hepatopancreas, among shrimps collected from various farms in the south and captured ones from Persian Gulf, even ones without clinical signs. ROI among species estimated, using the NATIVIDAD & LIGHTNER formula(1992b) and SOI were graded, using a generalized scheme for assigning a numerical qualitative value to severity grade of infection which was provided by LIGHTNER(1996), in consideration to histopathology and counting specific inclusion bodies in different stages(were modified by B. Gholamhoseini). Samples with clinical signs, showed grades more than 2. Most of the P. semisulcatus and M. affinis samples showed grade of 3, in the other hand in most of P. styliferus samples grade of 4 were observed, which can suggest different sensitivity of different species. All samples were tested by Nested PCR method with IQTm 2000 WSSV kit and 183 of 240 samples were positive and 3 1evel of infection which was shown in this PCR confirmed our SOI grades, but they were more specified.

Real-Time PCR Assays for the Detection of Puccinia psidii

Puccinia psidii (Myrtle rust) is an emerging pathogen that has a wide host range in the Myrtaceae family; it continues to show an increase in geographic range and is considered to be a significant threat to Myrtaceae plants worldwide. In this study, we describe the development and validation of three novel real-time polymerase reaction (qPCR) assays using ribosomal DNA and β-tubulin gene sequences to detect P. psidii. All qPCR assays were able to detect P. psidii DNA extracted from urediniospores and from infected plants, including asymptomatic leaf tissues. Depending on the gene target, qPCR was able to detect down to 0.011 pg of P. psidii DNA. The most optimum qPCR assay was shown to be highly specific, repeatable, and reproducible following testing using different qPCR reagents and real-time PCR platforms in different laboratories. In addition, a duplex qPCR assay was developed to allow coamplification of the cytochrome oxidase gene from host plants for use as an internal PCR control. The most optimum qPCR assay proved to be faster and more sensitive than the previously published nested PCR assay and will be particularly useful for high-throughput testing and to detect P. psidii at the early stages of infection, before the development of sporulating rust pustules.

Detección de Leptospira spp. en orina de caninos expuestos a factores de riesgo mediante la utilización de la técnica molecular pcr

Tesis (Magister en Ciencias Veterinarias).-- Universidad de La Salle. Facultad de Ciencias Agropecuarias. Maestría en Ciencias Veterinarias, 2014

A more reliable PCR for detection of Mycobacterium tuberculosis in clinical samples

Diagnostic techniques based on PCR have two major problems: false-positive reactions due to contamination with DNA fragments from previous PCRs (amplicons) and false-negative reactions caused by inhibitors that interfere with the PCR. We have improved our previously reported PCR based on the amplification of a fragment of the Mycobacterium tuberculosis complex-specific insertion element IS6110 with respect to both problems. False-positive reactions caused by amplicon contamination were prevented by the use of uracil-N-glycosylase and dUTP instead of dTTP. We selected a new set of primers outside the region spanned by the formerly used primers to avoid false-positive reactions caused by dTTP-containing amplicons still present in the laboratory. With this new primer set, 16 copies of the IS6110 insertion element, the equivalent of two bacteria, could be amplified 10(10) times in 40 cycles, resulting in a mean efficiency of 77% per cycle. To detect the presence of inhibitors of the Taq polymerase, which may cause false-negative reactions, part of each sample was spiked with M. tuberculosis DNA. The DNA purification method using guanidinium thiocyanate and diatoms effectively removed most or all inhibitors of the PCR. However, this was not suitable for blood samples, for which we developed a proteinase K treatment followed by phenol-chloroform extraction. This method permitted detection of 20 M. tuberculosis bacteria per ml of whole blood. Various laboratory procedures were introduced to reduce failure or inhibition of PCR and avoid DNA cross contamination. We have tested 218 different clinical specimens obtained from patients suspected of having tuberculosis. The samples included sputum (n=145), tissue biopsy samples (n=25), cerebrospinal fluid (n=15), blood (n=14), pleural fluid (n=9), feces, (n=7), fluid from fistulae (n=2), and pus from a wound (n=1). The results obtained by PCR were consistent with those obtained with culture, which is the "gold standard." We demonstrate that PCR is a useful technique for the rapid diagnosis of tuberculosis at various sites.

Population genetic structure of Neogobius caspius (Eichwald ,1831) in the South Caspian Sea using PCR-RFLP Marker

Neogobius caspius is a small benthic fish that is native to the Caspian Sea. The importance of this fish is because of it is role as a main food resource of the sturgeon fish. The genetic diversity of N. caspius population in the Caspian Sea was studied using PCR- RFLP technique. A total of 135 samples of N. caspius were collected from coastal line in the north Caspian sea, including specimens from coasts of Anzali , Torkman Port and Chalus. Genomic DNA was extracted by phenol-chloroform method and then was amplified using a pair primer of cytochrom b gene, 2 tRNA gene and the control region sequences by a thermal cycler. D2 (5'-CCGGAGTATGTAGGGCATTCTCAC-3'), CY1 (5'-YYTAACCRRGACYAATGACTTGA-3') 12 restriction enzyme were used to digest the target gene region including: Alul HincII —Tas1 —Rsa1 -MboI -DraI -BSeNI(BSRI) Alw261(BsmAI). Bsul 51 Hin11 Bsh12851- BsuRI(HaeIII) digested PCR products were observed by silver staining method followed by Polyacrylamide gel electrophoresis (PAGE). The results were shown the same pattern among the species. There was no polymorphism and no differentiation in population in the Neogobius caspius fish and all individuals have shown homogenous genotype.