Conventional Polymerase Chain Reaction Technique Research Articles

IDENTIFICATION OF MICROFILARIAE USING CONVENTIONAL POLYMERASE CHAIN REACTION AND QPCR-HRM

Background: The presence of filarial worms in the lymph nodes can result in acute symptoms, such as inflammation of the lymph nodes and ducts, particularly in the groin region. As part of the life cycle of filariasis, symptomatic or asymptomatic patients with microfilariae in their blood can transmit the disease via mosquito bites. The inspection of microfilariae that is currently being developed uses Polymerase Chain Reaction (PCR) to carry out a unique DNA search technique. Purpose: Identify the type of microfilaria present in filariasis patients using Quantitative PCR High- Resolution Melting (qPCR-HRM) and conventional PCR techniques. Method: This study involved the examination of 19 samples using the qPCR-HRM method. Subsequently, the results that were considered positive for microfilaria underwent further testing using conventional PCR. Result: The results of the examination using these two methods revealed the presence of Brugia malayi and Wuchereria bancrofti microfilariae with peak melting temperatures ranging from 78.2 – 78.7 °C and 80.8 – 81.2 °C, and fragment sizes of 199 bp and 227 bp, respectively. Conclusion: Based on the results of the identification from these two methods, it is evident that microfilariae of Brugia malayi and Wuchereria bancrofti can be detected using both conventional and qPCR-HRM methods.

Low-cost conventional PCR techniques enable simultaneous detection of bacterial sexually transmitted infections with enhanced sensitivity and specificity

PurposeNeisseria gonorrhoeae (NG), Chlamydia trachomatis (CT) and Mycoplasma hominis (MH), the three most common treatable bacterial sexually transmitted infections (STIs) worldwide can lead to many complications if remain untreated. Screening of high-risk population with highly sensitive methods will lead to significant improvement in patient outcomes and will prevent downward transmission. The advantages of Polymerase chain reaction (PCR) based assay are not only high sensitivity and specificity, but also detection of multiple organisms in a single reaction which reduce the result turn-around time. The aim of the present study was to evaluate the feasibility of a multiplex PCR assay method targeting 16S rRNA gene for simultaneous detection of NG, CT and MH infection along with their trend and occurrence among high-risk population in Assam, Northeast India. MethodsA cross-sectional study was undertaken, where a total of 200 randomly selected patients from high-risk population were included. After validation of singleplex PCR, Multiplex PCR (M-PCR) was performed along with the traditional culture method for NG. Results & conclusionThe overall agreement of M-PCR with singleplex PCR was very high (100%). The occurrence of STI was found to be very high (101/200; 50.5%). Furthermore, co-infection was detected in 10/200; 5%) individuals. Infection was more common among young individuals (p < 0.05) and males out-numbered females (p < 0.05). The most common organism detected was CT (42/200; 21%) followed by NG (41/200; 20.5%) and MH (20/200; 10%). The M-PCR assay workflow is simple, cost effective and can be used in routine diagnostic laboratories with basic molecular facilities.

Six-Year Study on Cutaneous Leishmaniasis in Al-Muthanna, Iraq: Molecular Identification Using ITS1 Gene Sequencing.

The current study aimed to determine the prevalence of cutaneous leishmaniasis (CL) in Al-Muthanna province (Iraq) and to characterize the Leishmania species that cause cutaneous lesions through conventional polymerase chain reaction techniques in some patients during the first 7 months of the year 2020. Medical information on patients with CL was obtained from archived records at the Al-Muthanna Health Office's Public Health Department (2015-2020). In the Al-Hussein Teaching Hospital laboratory, 95 CL samples were collected and examined microscopically for molecular characterization using Giemsa staining. Between 2015 and 2020, 2,325 patients (1,184 men and 1,141 women) were enrolled. Although CL occurred across all age groups, those aged range of 5-14 years had the highest proportion of infections (53.0%). This study found that most infections occurred between December and February, peaking in January. Only 63 of 95 CL samples were positive for the Internal Transcribed Spacer 1 region. L. tropica was found in 39 samples (61.9%), whereas L. major was found in 24 samples (38.1%), in CL patients. Although dermal lesions develop in all body regions, a single lesion is the most common. The upper limbs (13 of 16 samples, 33.3%)were infected with L. tropica, whereas the lower limbs (9 of 14 samples, 37.5%) were infected with L. major. In contrast to L. major, most L. tropica lesions occur in urban areas. Our study indicates that CL is endemic in the Al-Muthanna province and that two Leishmania spp. coexist in the province. Molecular diagnosis is a vital component in determining many clinical symptoms of the Leishmania parasite as well as implementing suitable therapeutic, epidemiological, and control strategies.

Evaluation of loop-mediated isothermal amplification method for efficient detection of the periodontopathic bacteria Porphyromonas gingivalis.

Periodontitis is a multifactorial, polymicrobial oral inflammatory illness brought on by oral pathogens. Porphyromonas gingivalis is a Gram-negative, obligatory anaerobic, black-pigmented coccobacillus and is regarded as a primary etiological factor in the progression of periodontitis. Rapid, highly senstitive and specific detection methods are emerging. The present study aimed to evaluate the loop-mediated isothermal amplification (LAMP) technique for efficiently detecting P. gingivalis from subgingival plaque samples of chronic periodontitis patients. This study included 50 subgingival plaque samples from patients suffering from chronic periodontitis. The DNA (Deoxyribonucleic acid) was extracted by the "modified proteinase K" method. A set of six primers, targeting the pepO gene of P. gingivalis, was used for conducting LAMP. The amplification was visualized by naked-eye detection and agarose electrophoresis. Conventional polymerase chain reaction (PCR) and real-time qantitative PCR (qPCR) were carried out by targeting the 16SrRNA (16S ribosomal ribonucleic acid) gene of P. gingivalis. The results showed that LAMP detected P. gingivalis in 40 out of 50 samples (80%). Whereas, qPCR and conventional PCR technique detected P. gingivalis in 38 (76%) and 33 (66%) samples respectively. The sensitivity and specificity of the LAMP method were 94.87% and 90.90%, respectively. With qPCR, the sensitivity and specificity were found to be 92.30% and 81.81%, respectively, whereas, with conventional PCR, it was found to be 76.92% and 72.72%, respectively. LAMP is an efficient technique for quick, accurate, and reliable identification of P. gingivalis from subgingival plaque samples. The technique needs to be validated analytically, and further studies can be conducted by taking saliva and/or gingival crevicular fluid samples from periodontitis patients.

Detection of pulmonary candidiasis in immunocopromasied Iraq patients by conventional polymerase chain reaction technique

Candida species was possible to cause pulmonary candidiasis, this infection commonly occurred in" immunocompromised patients. This study aimed to isolate and diagnose of pulmonary candidiasis by using molecular method (Conventional PCR). This study was included a total of hundred and fifty (n=150) samples of bronchoalveolar lavage were collected from 100 immunocompromised patients with underlying diverse diseases and control group included 50 bronchoalveolar lavage from immunocompetent individuals were composed from in-and out patients who attended of Al-Imammian Al-Kadhmain Medical City, Baghdad teaching Hospital, Baghdad, during the period from May 2014 to March 2015. Samples were cultured on CHROMagar and after that detected by using API 20 Candida stripe for initial identification of Candida isolates. Germ tube test, chlamydospore formation were also performed to reinforce identification them DNA isolates was extracted for conventional polymerase chain reaction (PCR) method. Results showed that all apparently healthy individuals samples were negative by conventional PCR. Concerning immunocompromised patients with underlying different diseases, 60 out of 100 samples revealed negative results by each of conventional and molecular methods Only 38 (38%) immunocompromised patients had positive results by molecular and conventional methods and two samples (2%) positive result by molecular methods also "show that CHROMagar was a good tool for initial identification of Candida isolates. This study concludes that candidasis may be as opportunistic infection among immunocompromised patients and PCR method confirmed more suitable diagnosed other than culture on CHROMagar medium and other phenotypic technique".

Frequency of OXA-Type Carbapenemases among Carbapenem-Resistant Acinetobacter baumannii in Clinical Isolates from Adult Intensive Care Unit in India

Abstract Purpose Acinetobacter baumannii is a highly virulent bacteria in modern health care, with a high ability to acquire antimicrobial resistance. Carbapenemases production appears to be the most common mechanism involved in drug resistance to carbapenem. As the prevalence of carbapenem-resistant A. baumannii was high in intensive care unit (ICU) patients, this study was designed to find the frequency of oxacillinases (OXA) genes including OXA-23, OXA-24, OXA-51, and OXA-58. Materials and Methods A clinical specimen was collected from patients admitted to the adult ICU. DNA was isolated from carbapenem-resistant A. baumannii and amplified using conventional polymerase chain reaction technique and gel electrophoresis for visualization of results. Results The frequency of the OXA-23 gene was high with 87.5%, followed by OXA-51 gene with 73.2%. All 56 isolates were negative for the OXA-24 and OXA-58 genes. We also found that both OXA-23 and OXA-51 genes coexisted in 40 (71.4%) isolates. No significant difference was found between drug-resistant genes (OXA-23 and OXA-51) and clinical outcomes. The relationship between the presence of OXA gene was compared between survivors and nonsurvivors, which was found out to be nonsignificant. The presence of OXA genes showed no significant increase in the length of hospital stay. The significant association between acute physiology and chronic health evaluation IV scores and clinical outcome was calculated, and it was evident in the comparison of the discharged and died groups. Conclusion Early detection of these drug-resistant genes by molecular methods is essential in decreasing the spread of carbapenem-resistant A. baumannii.

Molecular detection of Spirochetes and Borrelia burgdorferi in stray dogs of Nineveh province, Iraq.

Borrelia burgdorferi is a Gram-negative bacterium that causes Lyme disease or borreliosis in domestic and wild animals, including dogs, with the possible transmission to humans. This study was conducted to investigate the infection rate of Spirochetes and B. burgdorferi in stray dogs in Nineveh province, Iraq. During the period from May to October (2022), a total of 55 stray dogs were selected randomly from different areas in Nineveh province, Iraq. Blood samples were collected from cephalic venous and tested molecularly using the conventional polymerase chain reaction technique. The present study revealed that the total infection rates of Spirochetes and B. burgdorferi were 41.82% and 27.27%, respectively. Concerning age, values of infection rate, odds ratio, and relative risk of B. burgdorferi were increased significantly in dogs aged ? 4 months (42.86%, 3.505%, and 2.438%, respectively), while decreased in dogs of ? 1-3 (12.5%, 0.337% and 0.42%, respectively) and ? 3 (13.33%, 0.32% and 0.409%) years old when compared to dogs aged 5-12 months (27.27%, 1% and 1%, respectively). While concerning dogs sex, a significantly higher infection rate, odds ratio, and relative risk of B. burgdorferi were shown in females (32.56%, 5.495% and 6.792%, respectively) compared to males (8.33%, 0.182% and 0.147%, respectively). To the best of our knowledge, this represents the first Iraqi study on the prevalence of spirochetes, in particular B. burgdorferi, in stray dogs in Nineveh province (Iraq). However, additional studies of B. burgdorferi infection in other animals as well as vectors such as ticks in different geographic areas, appear necessary to detect variation in the distribution patterns of infection. In addition, owners and veterinarians should be aware of zoonotic diseases transmitted from wild and domestic animals, in particular those with tick-bite histories.

Prevalence of ovine theileriosis in Mosul city, Iraq

The present study aimed to determine the prevalence of ovine theileriosis (OT) in sheep in Mosul city, Iraq using microscopic examination (ME) of the blood smears stained with MGG- Quick stain and conventional polymerase chain reaction technique (c-PCR) to compare between c-PCR technique and ME as techniques for the diagnosis of disease, and to investigate the pattern and type of infections based on multiplex polymerase chain reaction technique (m-PCR). From October 2021 to May 2022, one-handed eighty-five Blood samples were drawn randomly from sheep in various regions of Mosul city. The overall prevalence of OT was 42% (22.7 out of 185) and 52.4% (97 out of 185) using microscopic examination and c-PCR technique, respectively. A slight agreement was observed between ME of blood smears and c-PCR technique according to Kappa value 0.190, with low sensitivity, specificity, and accuracy of ME method was 30%, 88.6%, 58.4%, respectively, compared with c-PCR technique. The prevalence of mixed infection 22.7% and single infection with T. lestoquardi 20% were significantly higher (P<0.05) than single infection with T. ovis 9.7%. This study concludes that OT is widespread in Mosul city, Iraq, and the c-PCR technique is more reliable and suitable for detecting Theileriainfection in sheep than the ME method.

THE PREVALENCE OF BLANDM, BLAVIM GENES AMONG ENTEROBACTER CLOACAE BACTERIA

This study was aimed to detect the prevalence of blaVIM and blaNDM genes among Enterobacter cloacae isolates that were isolated from Iraqi patients. About 50 bacterial isolates were collected from different hospitals in Baghdad city and all these isolates were diagnosed using biochemical tests and CHROMagar culture media and conformed using the Vitek II system. The antibiotic susceptibility for E.cloacae isolates was determined using the disk diffusion (Kirby Bauer) method and the results showed that these bacteria were showed resistance to Cefepime and Meropenem antibiotics in percentage (50%), (40%) respectively. In addition it is resistant to Amikacin, ampicillin/sulbactam, Piperacillin, and aztreonam was (20%), (50%), (40%), and (55%) respectively. Also it has been noted that all isolates showed resistance to Cefixime in percentage (100%). EDTA combined disc test was used to detect the ability of E. cloacae isolates to produce carbapenemase and the results were showed that only 8 isolates were gave positive results , While the prevalence of blaVIM and blaNDM was determined using the conventional Polymerase chain reaction technique. And the result showed that only four isolates harbored blaNDM while blaVIM was present in 3 isolates

Morphometric and molecular characterization of Moniezia species in sheep in Mosul city, Iraq

The current study examined 100 small intestines collected randomly from sheep slaughtered in the abattoir and butcher’s shops from different Mosul city / Iraq areas of both sexes (55 females, 45 males) and different ages. Moniezia expansa was diagnosed in 9 samples of intestines by studying the morphometric characteristics of these tapeworms, especially the mature segments, in which both the ovaries and vitelline glands appeared in the ring shape on either side of the body segments and the rosette-like shape of the interproglotidial glands. No significant difference was noticed between males and females of sheep in our study, and the infection rate was 10% in sheep less than a year old and older than two years, with no significant difference between the age groups. The results of the molecular analysis by using conventional polymerase chain reaction technique confirmed the diagnosis of these worms, which belong to the genus Moniezia, with a product reaction of 700 base pairs. The sequencing result shows two strains of Moniezia expanza, which isolated from Iraq (Moniezia expansa-Iraqi one and Moniezia expansa-Iraqi 2) were similar to each other had a significant distance to other strains. The study also showed that Moniezia expansa is different from the same species in other countries.

Diagnostic study of trypanosomiasis of cats in Mosul, Iraq.

Trypanosomiasis is a zoonotic parasitic disease endemic in Iraq but with limited information about its occurrence in cats. This study was designed to detect Trypanosoma spp. in cats using microscopic examination by Giemsa stain and conventional polymerase chain reaction (PCR) technique in Mosul, Iraq. A total of 120 blood samples from cats were microscopically examined using Giemsa stain. Only 35 positive blood samples were examined by the conventional PCR technique. Hematological changes were also reported. The infection rate of Trypanosoma spp. was 34.2% (41 out of 120). Results of conventional PCR technique for the positive 35 blood samples indicated 31.4% as Trypanosoma spp. and 20% Trypanosoma evansi. This study showed that the infection in younger cats was significantly more than in older cats, with significant differences between females and males. Affected cats suffered from fever, dullness, pale mucous membranes, emaciation, muco-purulent ocular discharge, anorexia, incoordination, and anemia. Results of the blood picture indicated increase in total leukocyte count and decrease in hemoglobin concentration, packed cell volume, and total red blood cells. Trypanosoma spp. and T. evansi infection in Mosul of Iraq is reported for the first time in cats, and younger cats were more affected than older cats.

The Prevalence and Prognostic Impacts of Nucleophosmin Mutations in Adult Patients with De Novo Acute Myeloid Leukemia

BACKGROUND: Acute myeloid leukemia (AML) is known as cancer of the blood and bone marrow (BM) and is regarded as the commonest acute leukemia in adult patients. AIM: In this study, the aim to investigate the nucleophosmin mutations and their prognostic impacts in patients that were recently diagnosed with AML. METHODS: We have included patients who were newly diagnosed with AML and presented to the medical oncology clinics, National Cancer Institute, Cairo University, during the period from August 2016 to December 2018. To assess the laboratory and hematological outcomes of our patients, total RNA was extracted from BM and converted to cDNA then the expression of nucleophosmin 1 (NPM1) type A mutation was done by real-time quantitative polymerase chain reaction (PCR). Comparative analysis was also conducted to study outcomes between the gene mutation groups. RESULTS: We have included 89 AML patients in our study with a median age of 43 years (18–77). NPM1 gene mutation was detected in 37.1% of our patients by conventional PCR technique and agarose gel electrophoresis, of which 18% were NPM1 type A mutation. No significant differences were noticed between our patients based on their NPM1 gene mutation status (wild and mutant) in terms of sex, hepatomegaly, splenomegaly, and complete remission (CR). Lymphadenopathy was the only significant factor (p = 0.023). Surprisingly we found 9/33 patients had NPM1 mutation with recurrent cytogenetic abnormality. We found no statistical significance between mutation A and mutation non-A groups in any of the studied outcomes as sex, clinical and laboratory data, and CR. CONCLUSION: NPM1 gene mutation A was relatively low among our population but did not significantly affect the outcomes.

The performance of the recombinase polymerase amplification test for detecting Leishmania deoxyribonucleic acid from skin lesions of patients with clinical or epidemiological suspicion of cutaneous leishmaniasis.

The diagnosis of cutaneous leishmaniasis (CL) is based on demonstration of the presence of the parasite in samples obtained from the lesions by direct examination (DE), culture or polymerase chain reaction (PCR)-based molecular tests. Recombinase polymerase amplification (RPA) represents an isothermal version of the conventional PCR (cPCR) technique, being ideal for detecting Leishmania DNA, especially in field conditions. A prospective and cross-sectional study was conducted to evaluate the diagnostic performance of RPA in the health centres of rural endemic sites or the evaluation centre (EC) of 11 Colombian municipalities and in a reference centre (RC). Samples of 128 patients with suspected CL were included and processed for analysis by RPA vs DE in the EC and RPA vs DE and cPCR in the RC. The RPA performed at the EC was more sensitive (90.4% [95% confidence interval {CI} 81.9 to 95.7]) than the DE (42-67%) and the specificity was 72.7% (95% CI 57.2 to 85.0). Both the sensitivity and specificity increased to 100% when adjusting by the imperfect reference standard analysis method. In the RC, the sensitivity of RPA vs cPCR was 72% and the specificity was 69.8%, while the sensitivity of cPCR vs the DE test was 78.8% and the specificity was 81%. The higher sensitivity and specificity shown by RPA in the EC, but also its ease and speed of use, justify performing RPA in the health centres of rural endemic sites. In addition, RPA eliminates the subjectivity inherent in the traditional DE.

Identification and Genetic Characterization of Mycoplasma Species Affecting Respiratory System in Egyptian Cattle

Abstract: Mycoplasma species are the main important causative agents causing pneumonia in cattle, there are about 200 species or more. The current work aimed to investigate various mycoplasma spp. isolated from the respiratory tract of cattle by microbial culture, conventional polymerase chain reaction technique, and target gene sequencing with phylogenetic analysis. An initial screening was done to confirm the presence of mycoplasma spp. by culture on PPLO’s agar, digitonin sensitivity and biochemical tests. 129 isolates were characterized by fried egg with depressed center colonies, digitonin sensitive and negative to glucose fermentation and arginine utilization test. Out of 305 samples, 20 samples were selected for amplification by PCR technique using Mycoplasma 16S rRNA primer. Seven samples were positive to Mycoplasma species and gave amplified band at 1013 bp. subsequently, the seven isolates were sequencing. Four sequenced isolates (EGS1, EGL6, EGL1 and EGS2) were closely related to each other and very close to Mycoplasma bovis strains and far from M. leachii 99/014/6. Two sequenced isolates (EGL2 and EGL5) were closely related to each other and very close to Mycoplasma bovirhinis strains and far from M. leachii 99/014/6. One sequenced isolate (EGL3) was very close to Mycoplasma arginini strain and far from M. bovis 99/014/6. From these results, we conclude that conventional culture methods for diagnosis either by isolation and identification of Mycoplasma is a time-consuming method to diagnose mycoplasma infection So, these methods can be replaced by PCR and genome analysis technology.

Detection of Trichomoniasis in cattle in Nineveh province

The current study was conducted on the detection of Tritrichomonas foetus in vaginal mucus of infected cows using conventional polymerase chain reaction technique (cPCR) in Nineveh province, Iraq. A total of 87 vaginal mucus samples were collected randomly from the vagina of Heifer cows of different ages (2-4, >4-6, >6 years old) and stages of pregnancy with different clinical status (early embryonic death, pyometra, abortion and healthy animals once) by washing cow’s vagina using artificial insemination pipette, DNA extraction of T. foetus was done from vaginal mucus samples, cPCR was attempt using TFR3 and TFR4 primers, Results indicated that 11 cows (12.6 %) were positive for T. foetus. The clinical status of cows demonstrated statistically significant (P<0.05) a higher percentage of Early Embryonic Death at (6.9%) compared to pyometra, abortion, and healthy cows. Furthermore, the percentage of T. foetusinfection was significantly (P<0.05) elevated among cows (>2-4 years old) at (8%) compared to (>4-6 years old) and (>6 years old) cows. This study concluded that T. foetusinfection was an elevated percentage of infection in cows with early embryonic death and in cows (>2-4) years old. This study is the first detected T. foetus in cattle in Nineveh province.

Loop-Mediated Isothermal Amplification (LAMP): Comparative Advances over Conventional PCR and Other Molecular Techniques

Gene amplification technology is essential in the fields of diagnostic medicine. The polymerase chain reaction (PCR) is central in the molecular studies and provides ways for diagnostic advancement in the areas. However, the requirement for thermal cycler in a dedicated facility for amplification of target genes in the PCR technique has been a bottleneck to many researchers. The limitations associated with PCR include cost implication, strict expertise necessity and relatively higher turn-around time. The emergence of loop-mediated isothermal amplification (LAMP) in the last two decades assists in bridging the undesirable gaps. This review aims to highlight the natural advantages of the LAMP technique over the existing conventional PCR and other isothermal molecular techniques. Available published articles on LAMP techniques reviewed, listed many outstanding advances of the method in comparison to traditional PCR technique. The mentioned advantages include simplicity, affordability, naked-eye result detection and many more. That made LAMP becomes a rapidly accepted method in the field of molecular diagnosis. Other essential features of LAMP in comparison with other emerging nucleic acid amplification techniques were adequately explained and presented in tabular form for research and quick reference purposes. Though LAMP has some few limitations, its advantages outweigh its flaws by filling the gap in the field of molecular biology diagnostics.

Heterogeneity: A stumbling block in the expression of human papillomaviruses 16 in oral squamous cell carcinoma

Background: Tobacco and smoking are the established risk factors for oral cancer, but studies have reported the occurrence of oral cancer, even in the absence of these established factors. This indicates the need to identify other possible risk factors. In recent years, viral carcinogens have gained interest in which human papillomaviruses (HPVs) are the most researched risk factor in oral carcinogenesis. Therefore, we conducted this study to evaluate the presence of HPV-16 in potentially malignant and malignant lesions of the oral cavity. Methods: The current study comprised 40 patients, and they were divided into two groups: Group A: 20 patients with histopathologically diagnosed oral potentially malignant disorders (leukoplakia/erythroplakia) and Group B: 20 patients with histopathologically diagnosed oral squamous cell carcinoma (OSCC). Tissue samples were collected from all patients and made into formalin-fixed paraffin-embedded (FFPE) blocks. Polymerase chain reaction (PCR) test was carried out to detect the presence of HPV-16. Results: In our study, we found that none of the FFPE tissue samples was positive for the HPV-16 genome using the conventional PCR technique. The level of significance (P value) was set at P

Estandarización de la técnica Real Time PCR para la detección molecular de la mutación V617F en el gen de JAK-2 en neoplasias mieloproliferativas crónicas

V617F mutation in exon 14 of Janus Kinase 2 gene (jak-2) is used as a molecular marker for the diagnosis of Philadelphia negative myeloproliferative neoplasms (Phi-) such as Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (MFP). To detect this mutation, we used conventional polymerase chain reaction technique (PCR), a simple and inexpensive technique, however, has some drawbacks that current technology allows to solve. During the last years, more sensitive molecular techniques have been incorporated in clinical practice to support the diagnosis, prognosis and follow-up of hematological patients. For its implementation in the clinical routine should be considered technical and economic aspects, so in this work, we evaluate the Real Time PCR technique as a diagnostic method for the detection of the Jak-2-V617F mutation, using in house primers design. Our result show that the technique implemented has a concordance index of 0.87 with the conventional PCR used in the molecular diagnosis of myeloproliferative neoplasms. In addition, it has the same specificity, greater sensitivity and, shorter execution time in relation to conventional PCR. The implementation of this diagnostic method in our Hospital is technically possible and commercially convenient.

Urinary circulating DNA and circulating antigen for diagnosis of schistosomiasis mansoni: a field study.

To evaluate three non-invasive assays for the diagnosis of schistosomiasis mansoni in an Egyptian village. Urine was collected for the detection of circulating cathodic antigen (CCA) and cell-free parasite DNA (cfpd) by Point-of-contact (POC)-cassette assay and PCR, respectively. These tests were compared to Kato-Katz (KK) faecal thick smear for detection of Schistosoma mansoni eggs. Disease prevalence by POC-CCA assay was 86%; by PCR it was 39% vs. 27% by KK. Compared to KK, the sensitivity of POC-CCA reached 100%, but its specificity was only 19.2% with 41% accuracy. Sensitivity of the PCR assay for cfpd was 55.56%, and specificity was 67.12% with 64% accuracy. A new end point was calculated for combined analysis of KK, POC-CCA assay and PCR. Sensitivity for the three tests was 52.94%, 90.2% and 76.47%; specificity was 100% for KK and PCR and 18.37% for POC-CCA. The accuracy calculated for the three tests at the end point was 76% for KK, 55% for POC-CCA assay and 88% for PCR. Conventional PCR assay for detection of cfpd provides a potential screening tool for intestinal schistosomiasis with reliable specificity, reasonable accuracy and affordable financial and technical cost.

Detecção de Campylobacter spp. em carcaças de frango de corte resfriadas e congeladas pelos métodos imunoenzimático, PCR e PCR em tempo real

ABSTRACT: In order to detect and identify Campylobacter spp. in broiler chicken carcasses, and to compare detection methods, 43 chilled and 43 frozen carcasses were collected and analyzed. Three methodologies were evaluated: an automated Enzyme Linked Fluorescent Assay (ELFA) VIDAS®30, Polymerase Chain Reaction (PCR) and real-time PCR. Only four chilled carcasses (4.6%) were considered positive for Campylobacter spp. by VIDAS®30 and no sample was positive when the conventional PCR technique was used. However, real-time PCR showed a higher incidence of contamination by Campylobacter spp. in broiler carcasses, with 45 (52.3%) positive samples. C. jejuni was the species most frequently reported in the samples (88.8%). No differences in the frequencies of Campylobacter spp. were observed between the chilled and frozen broiler carcasses. In conclusion, real-time PCR was the most sensitive method for the detection of Campylobacter spp. in chilled or frozen broiler carcasses, which were mainly contaminated by C. jejuni.