Semi-nested Polymerase Chain Reaction Technique Research Articles

A comparison of the efficiency of molecular and conventional methods in the diagnosis of cutaneous leishmaniosis.

Leishmaniosis is one of the most important vector borne diseases. Among different forms of the disease, cutaneous leishmaniosis (CL) is the most common. Determining the method of definitive diagnosis for the disease has been the aim of various studies. Therefore this study afforded an opportunity to investigate this subject. To diagnose CL in 150 suspected patients referred to Mehran and Dehloran health centers during June 2018 to November 2019, two polymerase chain reaction (PCR) methods were performed and compared with the in vitro culture and microscopic evaluation of stained slides. The smears were stained with Giemsa for microscopy and cultured in Novy-Nicolle-McNeal (NNN) blood agar for promastigote growth. For semi-nested PCR and PCR-RFLP, the tissue and serosity from the lesions were used for DNA extraction. The semi-nested PCR technique using minicircle kDNA gene showed the highest positivity rates among all diagnostic assays with 114/150 (76%) of the samples and was used as reference standard, followed by the PCR-RFLP test using ITS1 gene with 112/150, (74.7%) positivity rates, microscopy with 101/150 (67.3%) and then culture 72/150 (48%). microscopy and culture methods together improved overall positivity rates to 68.7% (103/150). The all positive samples using molecular technique were identified as Leishmania major. The highest sensitivity (98.3%), specificity (100%), accuracy (98.8%), negative predictive value (94.7%) and κ coefficient (0.96=almost perfect) was observed by comparing PCR-RFLP and semi-nested PCR. kDNA-semi-nested PCR and ITS1-PCR-RFLP presented an interesting alternative to conventional methods for the identification of CL and improved its diagnostic value significantly in suspected patients with negative direct smears.

Detection of Wuchereria bancrofti DNA in paired serum and urine samples using polymerase chain reaction-based systems.

The Global Program for the Elimination of Lymphatic Filariasis (GPELF) aims to eliminate this disease by the year 2020. However, the development of more specific and sensitive tests is important for the success of the GPELF. The present study aimed to standardise polymerase chain reaction (PCR)-based systems for the diagnosis of filariasis in serum and urine. Twenty paired biological urine and serum samples from individuals already known to be positive for Wuchereria bancrofti were collected during the day. Conventional PCR and semi-nested PCR assays were optimised. The detection limit of the technique for purified W. bancrofti DNA extracted from adult worms was 10 fg for the internal systems (WbF/Wb2) and 0.1 fg by using semi-nested PCR. The specificity of the primers was confirmed experimentally by amplification of 1 ng of purified genomic DNA from other species of parasites. Evaluation of the paired urine and serum samples by the semi-nested PCR technique indicated only two of the 20 tested individuals were positive, whereas the simple internal PCR system (WbF/Wb2), which has highly promising performance, revealed that all the patients were positive using both samples. This study successfully demonstrated the possibility of using the PCR technique on urine for the diagnosis of W. bancrofti infection.

Development of an efficient multiplex semi-nested PCR for convenient use in urine samples for diagnosis of toxoplasmosis

BACKGROUND: Toxoplasmosis is a protozoan parasitic infection. The cerebrospinal, ocular and congenital forms of the disease are complicated and life threatening. Diagnosis of toxoplasmosis is difficult due to invasive sample requirement, complications in pathogenesis, immunology, and interpretation of test results. Using urine as a non invasive sample source for molecular diagnosis of toxoplasmosis is the main object of this research. Attempts were made to diagnose toxoplasmosis using polymerase chain reaction (PCR) technique on the parasite’s deoxyribonucleic acid (DNA) in urine. METHODS: Parasite: The Toxoplasma gondii (T. gondii) RH strain was obtained from peritoneal exudates of small white laboratory mice inoculated with 5x105 tachyzoites three days before aspiration. Urine samples with defined numbers of tachyzoites per ml were used as laboratory samples. The target was a 529 bp segment (AF146527 gene bank) of the T. gondii genome with 200-300 repeats as target in PCR. Selected primers were designed on its sequence. RESULTS: A multiplex semi-nested PCR technique was developed for obtaining a method for precise diagnosis of toxoplasmosis, time and budget saving and to diminish the DNA cross contamination risk. It was sensitive to detect 2 this tachyzoites DNA per final sample. CONCLUSION: With further development, nested PCR can be a useful and non-invasive method for diagnosis of cerebrospinal, ocular, and congenital toxoplasmosis.

A non-stop, single-tube, semi-nested PCR technique for grading the severity of white spot syndrome virus infections in Penaeus monodon.

A single-tube, non-stop, semi-nested polymerase chain reaction (PCR) technique was developed for simultaneous detection and severity grading of white spot syndrome virus (WSSV) infections in the black tiger shrimp Penaeus monodon. The test uses 1 sense primer and 3 antisense primers that produce up to 3 PCR products (1100, 526 and 250 base pairs [bp]) depending upon the severity of infection. Specifically, heavy infections (> or = 2 x 10(4) viral particles) of WSSV produce all 3 fragments, while moderate infections (around 2 x 10(3) viral particles) produce 2 (526 and 250 bp) and light infections (20 to 200 viral particles) produce 1 (250 bp). In addition, the technique uses internal control primers that yield a shrimp characteristic fragment for non-infected samples and samples with a low quantity of viral target in order to assure integrity and reproducibility of the PCR assays. The non-stop, single-tube, semi-nested PCR technique is simple and convenient and can detect as little as 5 fg WSSV DNA (20 viral particles) in crude extracts of postlarval samples or extracts of pleopods and haemolymph from larger shrimp.

DETECTION AND QUANTITATIVE MEASUREMENT OF TRANSFORMING GROWTH FACTOR-β1 (TGF-β1) GENE EXPRESSION USING A SEMI-NESTED COMPETITIVE PCR ASSAY

A polymerase chain reaction (PCR) technique was optimized for detection and quantification of very low concentrations (down to a few molecules) of transforming growth factor β1 (TGF-β1) mRNA. The strategy involved a combination of a competitive PCR assay and a semi-nested PCR. In the present study, the semi-nested PCR technique was tested in several rat organs containing different concentrations of target mRNA. A control fragment for TGF-β1 was used to correct for differences in amplification of various cDNA samples. TGF-β1 mRNA levels were also corrected according to the abundance of the “housekeeping” gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in the same samples. The differences of sensitivity among the standard (one-step) and semi-nested (two-step) competitive PCR assays for the detection of TGF-β1 are discussed. In conclusion, the semi-nested PCR protocol provides greatly enhanced sensitivity over standard PCR analysis. It is a reproducible and very specific method for quantification of only a few molecules of TGF-β1 mRNA in a background of non-target molecules.

Assessment of Clonality in Cutaneous Lymphoid Infiltrates by Polymerase Chain Reaction Analysis of Immunoglobulin Heavy Chain Gene Rearrangement

Determination of the biologic potential of cutaneous lymphoid infiltrates may be difficult by standard histologic or immunohistologic examination. The polymerase chain reaction (PCR) has been used to document clonal rearrangements of the immunoglobulin heavy chain gene in paraffin-embedded fixed tissues. To explore the value of PCR in evaluation of cutaneous lymphoid infiltrates, 93 archival nonmycotic lymphoid lesions (28 small or mixed lymphocytic lymphomas, 15 large cell lymphomas, 40 benign infiltrates, and 10 with atypical features) were analyzed. These cases had been previously immunophenotyped on paraffin sections, and clinical follow-up from 7 to 20 years was gathered. DNA products were generated using a seminested PCR technique, separated by 5% polyacrylamide gel electrophoresis, stained with ethidium bromide, and visualized under UV light. Cases with a 100- to 120-base pair band were scored as positive. Of the 28 small or mixed cell lymphomas, 23 had a B-cell immunophenotype or consisted of a mixture of B and T cells; of these, seven (30%) demonstrated a monoclonal pattern, and three (13%) were indeterminate. Twelve large cell cases were B-cell or mixed; five (42%) of these were positive for a monoclonal band, while four (33%) were indeterminate. None of five T-cell small cell or three T-cell large cell lymphomas demonstrated a monoclonal band. In contrast, 39 of the 40 benign cases were T-cell predominant or mixed lesions. Nevertheless, 18 of these 40 cases on initial testing suggested possible monoclonality. Six were indeterminate, and 12 demonstrated apparent monoclonal bands, of which four were reproducible on repeat testing. No histologic or clinical features of lymphoma were present in 17 of these 18 cases, suggesting that they represent false-positive results. Most of the latter lesions showed sparse perivascular infiltrates, with very few B cells. This suggests that amplification of the immunoglobulin heavy chain gene from a small number of lymphocytes may produce a monoclonal band. In summary, PCR may provide adjunct information about clonality in selected lymphoid skin lesions, but is rather insensitive in this setting. Such data must be carefully considered in the context of the histologic, immunohistologic, and clinical findings, particularly when assessing sparse infiltrates, because of the potential for false-positive results.

Immunoglobulin gene rearrangements in lymphoplasmacytic infiltrates of labial salivary glands in Sjögren's syndrome: A possible predictor of lymphoma development

Sjögren's syndrome is an autoimmune disorder in which patients have a well-recognized risk of developing malignant lymphoma. Although some clinical parameters may herald the onset of lymphoma, few reliable histologic or molecular markers are available that predict progression to a malignant lymphoproliferative disorder. The purpose of this study was to identify the prevalence of immunoglobulin heavy chain monoclonality in labial gland biopsies of patients with Sjögren's syndrome and to compare this to clinical outcome. The polymerase chain reaction was applied to 76 sequential labial salivary gland biopsies from patients under investigation for Sjögren's syndrome. A seminested polymerase chain reaction technique was used on DNA extracted from formalin-fixed, paraffin-embedded tissue to amplify the V-D-J region of the immunoglobulin heavy chain gene. Thirty-four randomly selected labial salivary glands that showed nonspecific sialadenitis from patients without Sjögren's syndrome were used as controls. Monoclonality, as defined by a single band on polyacrylamide gel electrophoresis was detected in 11 cases (14.5%). Of cases that showed monoclonality, four patients were subsequently diagnosed with extrasalivary lymphoma. In each case the rearranged bands in the lip biopsy and the lymphoma were the same size. In one patient who later developed lymphoma, a monoclonal rearranged immunoglobulin band was not identified. In addition, no cases of the translocation t(14;18) were identified by polymerase chain reaction in any of the lip biopsies showing heavy chain monoclonality or in any of the extrasalivary gland lymphomas. These results suggest that monoclonal immunoglobulin heavy chain gene rearrangements are a relatively common finding in patients with Sjögren's syndrome and may prove to be a useful marker for predicting the progression to, and early detection of malignant lymphoma.

Detection of Monoclonal B-Cell Populations in Decalcified, Plastic-embedded Bone Marrow Biopsies with the Polymerase Chain Reaction

Polymerase chain reaction (PCR) has been employed successfully for the detection of clonal immunoglobulin gene rearrangements in paraffin-embedded clinical samples. The authors examined whether this technique can also be applied to fixed, decalcified, and plastic-embedded bone marrow biopsies. DNA extracted from 66 glycolmethacrylate-embedded trephine biopsy samples was amplified for the detection of rearranged VDJ regions of the immunoglobulin heavy chain genes using both a single-step and a semi-nested PCR technique. After exclusion of samples with inadequate DNA, clonality was confirmed in 16 (67%) of 24 cases with B cell malignancy, whereas all 11 non-B cell neoplasms, and 6 of 9 cases with normal bone marrow showed evidence of a polyclonal B cell population. Patterns indicating oligo- or monoclonality were observed in three plastic-embedded samples of normal bone marrow, although control PCR of frozen bone marrow samples obtained in parallel showed no evidence of clonality. Repeated PCR of these cases revealed inconsistent bands, probably due to the amplification of rare templates from polyclonal B cells. Decalcified, plastic-embedded bone marrow biopsies are suitable for PCR-based determination of B-cell clonality. To exclude the possibility of false-positive results, monitoring of template DNA quality and independent control amplifications are mandatory.

Detection of immunoglobulin gene rearrangement in lymphoid malignancies of B‐cell lineage by seminested polymerase chain reaction gene amplification

Seminested polymerase chain reaction (PCR) was used to amplify the DNA fragments of the complementarity-determining region 3 of the immunoglobulin (Ig) gene heavy chain from the malignant cell specimens of patients with leukemias and lymphomas of B-cell lineage. Two different pairs of primers were used sequentially. Twenty of the 27 (74%) acute lymphoblastic leukemia (ALL) patients, 14 of 19 (74%) chronic lymphocytic leukemia (CLL) patients and eight of 20 (40%) non-Hodgkin's lymphoma (NHL) patients, who had rearrangement of the Ig gene heavy chain by Southern analysis, were positive by the seminested PCR. False-negative results appeared to occur more commonly in cases of lymphoma. The PCR analysis was also less likely to be positive if one-stage PCR studies with either pair of primers were both negative. The seminested PCR technique was found to have a high sensitivity of detecting malignant cells at the level of 0.02%. The clinical application of this assay needs to be investigated further.