Nested Polymerase Chain Reaction Method Research Articles

A colorimetric assay for Neospora caninum utilizing the loop-mediated isothermal amplification technique

Neospora caninum (N. caninum) is a protozoan parasite that poses a serious risk to livestock by infecting various domestic and wild animals. Loop-Mediated Isothermal Amplification (LAMP) offers a cost-effective, highly sensitive, and specific method for detecting protozoan parasites. This study aims to develop a precise, rapid, and visually assessable colorimetric LAMP method, improving on traditional techniques. We employed a rigorous screening process to identify the optimal primer set for this experiment. Subsequently, we fine-tuned the LAMP reaction at 65 °C for 40 min with 270 μmol/L neutral red. We then confirmed the specificity of primers for N. caninum through experimental validation. The LAMP method demonstrated a lower detection limit compared to traditional Polymerase Chain Reaction (PCR) techniques. While LAMP offers clear advantages, the prevalence of DNA detected in 89 sheep serum and 59 bovine serum samples using the nested PCR method was 3.37 % (3/89) and 1.69 % (1/59), respectively. In contrast, when the LAMP method was employed, the prevalence of detected DNA rose to 5.61 % (5/89) for sheep and 3.38 % (2 /59) for bovine. A comparison of two molecular assays using the intragroup correlation coefficient (ICC) resulted in a value of 0.999 (95 % CI: 0.993–0.996, p < 0.001), indicating the LAMP method is in the “better” range according to James Lee's categorization. The LAMP technique, optimized with specific primers of N. caninum and neutral red dye, not only exhibited higher sensitivity but also provided convenience over conventional PCR methods, highlighting its potential for on-site applications and cost-effective field detection.

Molecular detection of Plasmodium DNA in goats in Kulon Progo, Indonesia

The Plasmodium parasites transmitted through the bites of infected Anopheles mosquitoes cause malaria in humans. Despite extensive research on the malaria parasite in ungulates, its occurrence in goats in Indonesia, particularly in the Kaligesing and Girimulyo border areas, has yet to be reported. This study aimed to investigate the presence of malaria parasites in goats from these regions. We collected 81 blood samples from goats of different ages and sexes. Blood samples were drawn from the jugular vein and preserved using EDTA tubes. We evaluated the overall clinical status of the goats by monitoring their rectal temperature and capillary refill time (CRT). Microscopic analysis was carried out by observing blood smears under a microscope, but the Plasmodium parasite was not detected. Next, we employed a molecular method to detect Plasmodium DNA using a nested polymerase chain reaction (PCR) method that targeted the cytochrome C oxidase subunit I (COI) gene. The results confirmed the presence of Plasmodium DNA in 5 out of the 81 samples tested. The obtained data were analyzed descriptively, and Wilson’s confidence interval method was used to calculate confidence limits for the sample proportions. This study provides the first molecular detection of previously undocumented malaria parasites in goats within the Girimulyo region of Kulon Progo, Yogyakarta, Indonesia. Identifying Plasmodium sp. using molecular techniques highlights the need for continued surveillance and management strategies to prevent the spread of malaria in animals and humans.

A New Duplication in 668 to 672 Sites of UL54 Gene in Cytomegalovirus Ganciclovir Resistant Liver Transplanted Patients

Background: In liver transplant recipients, human Cytomegalovirus (CMV) infection is a significant concern. Ganciclovir is the preferred medication for treating widespread CMV infections. In some cases, patients might require high doses of treatment for CMV infections that are resistant to ganciclovir, although liver transplant patients who have received extended ganciclovir and valganciclovir prophylaxis have reported infrequent cases of ganciclovir-resistant CMV infections. Mutations in the UL54 gene, responsible for encoding deoxyribonucleic acid (DNA) polymerase, can result in resistance. Objectives: In this study, the focus was on UL54 mutations and their association with ganciclovir resistance. Methods: In this study, 23 liver transplant recipients who were admitted to the transplant departments of Namazi and Abu Ali Sina hospitals within 2015 and 2017 were examined. Cytomegalovirus infection was then confirmed in them using the quantitative real-time polymerase chain reaction (PCR) method. The UL54 mutations were found after electrophoresis using the nested PCR method, and the PCR products were subsequently sequenced using the Sanger method. Sequence analysis and locating of UL54 mutations were performed using Finch software (version 1.4.0). Results: After sequencing 52 samples from 23 patients, 25 mutations in the UL54 gene were identified in 9 patients who were CMV-infected, occurring at a median of 32 days after transplant. These mutations, including S655L (10/9, 40%), N685S (8/9, 32%), F669L (4/9, 16%), A688V (2/9, 8%), and the novel AK124703.1: p.V668-G672dup (1/9, 4%), were detected in 9 liver transplant recipients over a median period of 2 years in the UL54 gene. Furthermore, a phylogenetic analysis was conducted to investigate the origins of these mutations in CMV isolated from the Iranian population. Conclusions: Considering that treatment with the drug ganciclovir has led to resistance mutations, particularly the new AK124703.1:p.V668-G672dup mutation, and inefficiency in treatment, it is necessary to determine drug-resistant CMV strains and closely monitor these patients. This includes determining viral load, assessing response to treatment, and identifying non-response at regular intervals until the viral load is completely eradicated in order to ensure the effectiveness of the treatment.

A Novel Nested Multiplex Polymerase Chain Reaction Assay for Malaria Diagnosis Using the Hydroxymethyl Dihydropterin Pyrophosphokinase-Dihydropteroate Synthase (hppk-dhps) Gene.

There are many techniques for malaria diagnosis. Currently, the nested polymerase chain reaction (PCR) method based on a small subunit ribosomal RNA gene (18S rRNA) has been used as a confirmatory method. However, this method is time-consuming, laborious, and costly. Therefore, the objective of this study was to develop nested multiplex PCR for Plasmodium species identification using the dihydropterin pyrophosphokinase-dihydropteroate synthase (hppk-dhps) gene. Genus- and species-specific primers for the hppk-dhps gene were designed. The performance of the novel nested multiplex PCR was compared with 18S rRNA nested PCR. A total of 115 blood samples were used in this study, including 84 infected samples and 31 uninfected samples. Analysis of the blood samples by nested multiplex PCR targeting the hppk-dhps gene identified 81 infected cases. The level of agreement between this novel method and 18S rRNA nested PCR was 97.4%. Further, the novel method successfully detected all human malaria parasites except Plasmodium ovale and detected mixed Plasmodium falciparum/Plasmodium vivax infections. The sensitivity and specificity obtained from this novel method were 96.4% and 100%, respectively. The limit of detection of the hppk-dhps nested multiplex PCR for P. falciparum and P. vivax was 500 parasites/µL and 4 parasites/µL, respectively. The lowest parasite gDNA detected by this method was 0.5 ng/µL for P. falciparum and 0.1 ng/µL for P. vivax. These results corroborate that the hppk-dhps gene is a novel amplification target for the detection of human malaria. This novel target PCR-based method is a beneficial approach for malaria diagnosis, as well as species identification and differentiation.

Molecular Detection of Borrelia spp. in Ticks of Sheep and Goats by Nested PCR Method in West Azerbaijan Province, Iran.

Background: Ticks are an important vector among arthropods associated with serious medical and veterinary problems. In this research, we investigate Borrelia species in ticks isolated from the surface of livestock (sheep and goat) in different regions of West Azerbaijan province. Materials and Methods: Polymerase chain reaction (PCR) was performed using specific primers targeting Borrelia spp. genes. Borrelia spp. was identified through PCR. The positive PCR products were sent to Pishgam Company for sequencing. Sequenced data were analyzed, and phylogenetic analysis was performed using maximum likelihood method in MEGA V.10. Results: The detection rate of Borrelia spp. for 16srRNA gene 69 (n = 542; 12.7%; 95%Cl: 10.1%-15.8%), 42 (n = 542; 7.7%; 95%Cl: 5.78%-10.1%) positive on 5S-23SrRNA gene and ospA gene 4 (n = 542; 0.74%; 95%Cl: 0.29%-1.88%). Conclusion: These results are the first report in Iran to identify Borrelia spp. These results of the study showed that the Borrelia spp. in hard ticks detected by PCR and it was negative to soft ticks. it is important in public health implications at the studied areas.

High Frequency of HPV Genotypes 16 and 18 Found in Breast Cancer Patients: Evidence for a More Comprehensive HPV Vaccination Program in Iran

Background &amp; Objectives: Papillomaviruses are found in many different types of infections and in a wide range of animals and humans. They can cause health problems, including benign and malignant tumors. In the present study, the association between human papillomavirus (HPV) infection and breast cancer (BC) in Iran was investigated.&#x0D; Materials &amp; Methods: In this cross-sectional study, the presence of the HPV genome was investigated in BC-suspected tissues for the first time in Qom Province, Iran. A total of 400 samples (including 200 BC-suspected tissue samples and 200 blood samples of women without BC) were collected from women referred to two cancer-specific general hospitals. To determine the presence of the L1 gene of HPV in the collected samples, nested polymerase chain reaction (PCR) was performed. Then, HPV-positive samples were tested by PCR using high-risk specific HPV-16 and 18 primers.&#x0D; Results: Out of 200 BC-suspected tissue samples, 172 were malignant (in terms of pathology). Based on the nested PCR method, the L1 gene of HPV was detected in 12% (24/200) of the BC-suspected tissue samples and in 1.5% (3/200) of the blood samples from women without BC. The high-risk HPV genotypes (which were the predominant types) were present in 75% of the samples.&#x0D; Conclusion: The results of the current study show a high frequency of HPV-16 and 18 genotypes in human BC in Iranian women. This is almost certainly due to poor rates of HPV vaccination, and it is strongly recommended that health organizations (such as the World Health Organization [WHO]) ensure adequate coverage of highly effective HPV vaccination in Iran.

Detection of "Candidatus Liberibacter asiaticus" Using Conventional PCR and Nested-PCR and Identification of Primer Sensitivity through Q-PCR in HLB Infected Mandarin Orange and Acid Lime Plants in Tamil Nadu

One of the most destructive citrus diseases in the world is called citrus Huanglongbing (HLB). For effective HLB associated ‘Candidatus liberibacter spp.’ prevention, sensitive and precise diagnostics are essential. Candidatus Liberibacter asiaticus (CLas), Candidatus Liberibacter africanus (CLaf), and Candidatus Liberibacter americanus (CLam) are among the ‘Candidatus liberibacter spp.’ that infect citrus. The most common of these is the species known as Las. To assess the impact of citrus greening disease in the citrus orchard, leaf samples exhibiting greening symptoms were picked up from 210 mandarin orange trees from nine localities in four districts and among them 189 plants showed HLB positive and leaf samples are picked up from 216 acid lime trees from nine localities in six districts of Tamil Nadu where citrus is cultivated in a larger area and among them 202 plants showed HLB positive. A “conventional singleplex polymerase chain reaction (PCR) Nested PCR and Q-PCR (Quantitative PCR)” was performed on the extracted DNA samples to detect Ca. Liberibacter asiaticus. Among all the mandarin orange growing localities from “Kanalkadu and Thandikudi in Dindigul district” and among the acid lime fields, the samples from “Sankarankoil from Tirunelveli district and Kallar from Nilgiris district” showed consistent amplification of 1160 base pairs fragment by using specific primers OI1 and OI2C and also 703 base pairs fragment with gene-specific primers A2 and J5 targeting beta-operon (rpiKAJL-rpoBC) gene which is a ribosomal protein gene of Ca. Liberibacter asiaticus, these results showed that these districts are exclusively positive to Clas . The nested PCR method significantly increased the sensitivity to identify Las up to 10 times and 100 times, respectively, compared to qPCR and conventional PCR in this study. A set of nested PCR primer pairs were examined to diagnose Las. Nested PCR was used to analyse 14 samples from 2 different citrus cultivars in 2 different districts. Among those again kanalkadu orchard for mandarin orange and Sankankoil orchard for acid lime showed more disease incidence and consistent amplification. The findings indicate that all the samples tested positive for HLB; the samples of blotched and chlorotic leaves from the acid lime field at Sankarankoil (thirunelveli district) and mandarin orange field at kanlakaadu (Dindigul district) had the highest positive detection rate (100%). The outcomes show that the nested PCR primer pairs are capable of detecting Las in a variety of symptomatic tissues, citrus cultivars, and geographical locations. The set of nested PCR primers used in the current work will be a very helpful addition to the existing methods for Las detection.

Assessment of Plasmodium falciparum Resistance to Artemisinin Among Attendees of Selected Health Facilities in Rivers State, Nigeria

Across-sectional research, aimed at evaluating artemisinin drug resistance in falciparum malaria patients attending selected health facilities in Rivers State, Nigeria, was done between July 2019 and March 2020. The selected hospitals were Rivers State University Teaching Hospital (RSUTH), University of Port Harcourt Teaching Hospital (UPTH) and Model Primary Health Centre (MPHC), Iriebe, all in Rivers State, Nigeria. The study population was 1000 subjects with an overall malaria prevalence of 25.5%, the number of enrolled participants from RSUTH was 494 with the highest infection rate of 32.5%, 300 from UPTH which had the least (11%) prevalence, while 206 were from MPHC, Iriebe with 29.6% prevalence accordingly. Blood samples were collected from these subjects by phlebotomy method. Plasmodium falciparum parasite was determined by microscopy using thick and thin blood films stained with Giemsa staining technique. Artemisinin resistance markers were assessed using the nested polymerase chain reaction method. The study population consisted of children, pregnant women, and other adults of both sexes. Out of the forty-seven isolates assayed for K13 and ATPase 6 genes, who carried the artemisinin-sensitive wild-type allele, none of these isolates exhibited both ATPase 6 and Kelch -13 resistance genes/mutations hence no single nucleotide polymorphism was observed. This suggests that this part of the country is not under the threat of artemisinin resistance. However, this should not undermine the need for continuous monitoring and surveillance of the response of P. falciparum to Artemisinin and Artemisinin combination therapy (ACTs) in River State.

Molecular Detection of Multiple Genotypes of Orientia tsutsugamushi Causing Scrub Typhus in Febrile Patients from Theni District, South India.

Scrub typhus (St) is a re-emerging mite-transmitted public health problem in Southeast Asia with escalating case incidences in the endemic areas. Though, more than 40 genotypes of the causative agent Orientia tsutsugamushi (Ot) have been documented, the information on the circulating genotypes in India is scanty. A hospital-based retrospective screening was undertaken to map the circulating molecular subtypes of the etiological agent in serologically confirmed scrub typhus (St) human cases, by targeting the GroEL gene of O. tsutsugamushi using the nested polymerase chain reaction method. Nine out of 34 samples (26%) yielded positive results and DNA sequencing analysis of six positive samples out of nine revealed that the sequences were related to three major genotypes, such as Karp (HSB1, FAR1), Kato (Wuj/2014, UT76), and Kawasaki (Kuroki, Boryong, Gilliam, and Hwasung). Additionally, the St-positive samples exhibited 100% and 99.45%; 97.53% and 97.81%; 96.99% nucleotide identity with the closely related Karp, Kato, and Kawasaki-related sequences, respectively. Overall, 94% of the nucleotides were conserved, and the variable site was 20/365 (5.5%). The prevalence of multiple genotypes among human cases further stresses the need to conduct in-depth studies to map the genotypes and their clinical relevance, and the contributing risk factors for the emergence of St cases in this area.

Estimating the Incidence Frequency of Helicobacter pylori Co-infection with Hepatitis C Virus among Thalassemia Individual in Wasit Governorate of Iraq

Objective(s): Helicobacter pylori (H. pylori) is a gram-negative bacterium that plays a significant role in developing gastrointestinal diseases such as peptic ulcers. H. pylori infection affects more than half of the world’s population. This pathogen incidence becomes more effective among thalassemia patients, a common genetic disorder characterized by a point mutation that leads to impaired production of the β-globin chain. Chronic hepatitis C is a chronic disease caused by the hepatitis C virus (HCV) that has significantly increased morbidity and mortality rates in these patients due to liver failure or hepatocellular carcinoma. Methods: Current study included 90 β-thalassemia patients admitted to the thalassemia hematology center in Al-Kut women and children Hospital, Wasit province, Iraq from September 2020 to November 2021. The patients are divided into two groups (β-thalassemia with HCV group and β-thalassemia without HCV). First and second groups contain 32 and 58 specimens of blood and stool samples, respectively. The patient diagnosed with thalassemia syndrome regularly attended the thalassemia hematology center for transfusion and chelation and follow up on Hb level and iron status. This study aimed at serological and molecular diagnosis of co-infection H. pylori with Hepatitis C virus (HCV) among thalassemia patients and diagnosis infection rate. Diagnosis was used Serological enzyme-linked immunoassay (ELISA) techniques for HP-IgG, Level of ferritin, liver function test will be determined and Nested PCR method targeting the molecular diagnosis will be used to confirm H. pylori 23S rRNA using DNA extracted from stool samples. Results: 90 β-thalassemia patients sample were diagnosed by Serological ELISA techniques for HP-IgG and the result showed 30 samples were positive (18 from the β-thalassemia with HCV group and 12 from the β-thalassemia without HCV)and 60 samples negative for HP-IgG and Nested polymerase chain reaction (PCR) method targeting the molecular diagnosis will be used to confirm H. pylori 23S rRNA using DNA extracted from stool for 30 stool samples revealed 13 samples positive (9 from the β-thalassemia with HCV and 4 from β-thalassemia without HCV) while 17 samples were negative. Conclusions: The predominance of H. pylori among patients in Wasit was 43.3%. Levels of ferritin have significant correlation with H. pylori in HCV patients. There was a positive correlation between hepatitis C status and high H. pylori rate when compared with β-thalassemia without HCV. Splenoctomy was a positive correlation with H. pylori in β-thalassemia with HCV. Moreover, these results recommend study about the virulence factor and genes. Other molecular studies are needed for detecting more virulence genes in H. pylori bacteria such as real-time PCR and sequencing of H. pylori in patients with thalassemia, especially those with high serum ferritin.

Development of a nested PCR assay for specific detection of Metschnikowia bicuspidata infecting Eriocheir sinensis.

In recent years, the “milky disease” caused by Metschnikowia bicuspidata has seriously affected the Eriocheir sinensis culture industry. Discovering and blocking the transmission route has become the key to controlling this disease. The existing polymerase chain reaction (PCR) detection technology for M. bicuspidata uses the ribosomal DNA (rDNA) sequence, but low sensitivity and specificity lead to frequent false detections. We developed a highly specific and sensitive nested PCR method to detect M. bicuspidata, by targeting the hyphally regulated cell wall protein (HYR) gene. This nested HYR-PCR produced a single clear band, showed no cross-reaction with other pathogens, and was superior to rDNA-PCR in specificity and sensitivity. The sensitivity of nested HYR-PCR (6.10 × 101 copies/μL) was greater than those of the large subunit ribosomal RNA gene (LSU rRNA; 6.03 × 104 copies/μL) and internal transcribed spacer (ITS; 6.74 × 105 copies/μL) PCRs. The nested HYR-PCR also showed a higher positivity rate (71.1%) than those obtained with LSU rRNA (16.7%) and ITS rDNA (24.4%). In conclusion, we developed a new nested HYR-PCR method for the specific and sensitive detection of M. bicuspidata infection. This will help to elucidate the transmission route of M. bicuspidata and to design effective management and control measures for M. bicuspidata disease.

Prevalence and diagnosis of retroviral infections in cats

Cat retroviruses, feline immunodeficiency virus and feline leukemia virus cause diseases that have a wide range of clinical manifestations and forms of infection. Aretrospective analysis of data from the last three years in the studied hospitals showed that the infection of cats with retroviruses is 32.6% for FeLV and 13.6% for FIV from the number of infectious diseases. Such high rates indicate that one third of cats coming to veterinary clinics with infectious diseases are infected with retroviruses. These are primarily animals at risk that have free range. Early detection of such animals by a veterinarian is important for effective treatment and prevention of infection. We have adapted the algorithm for the diagnosis of retroviral infections in cats to domestic conditions and developed practical recommendations for veterinarians on the effective choice of laboratory methods for the diagnosis of retroviral infections. It was found that in clinics, the initial study of whole blood samples from infectiously infected cats was performed using commercial serological test systems VetExpert FIVAb + FeLVAg. After the results of the first stage of the study, the protocol of the nested polymerase chain reaction (PCR) was tested. DNA isolation was performed using the IndiSpin Pathogen Kit. OneTaq® 2X Master PCR mix with Standard Buffer and specific oligonucleotides were used in the study. Proviral DNA was detected in 100% of blood samples for the respective pathogens FIV and FeLV. All animals that were pre-diagnosed with FIV and VLC infection at the first stage of diagnosis were diagnosed with proviral DNA and confirmed at the second stage of diagnosis. The use of the nested PCR method significantly increases the sensitivity and specificity of the study. The first stage of diagnosis allows a veterinarian to determine the infectious status of the animal on the basis of which to choose a treatment strategy and prevent transmission of the pathogen. The second stage of diagnosis allows you to clarify the previous diagnosis and establish the form of infection. The presence of a clear and understandable diagnostic algorithm is necessary for effective control of FIV and FeLV. Key words: polymerase chain reaction, feline viral leukemia, feline immunodeficiency virus, molecular diagnostics, retroviral infections, diagnostic algorithm.

Establishment of Noninvasive Methods for the Detection of Helicobacter pylori in Mongolian Gerbils and Application of Main Laboratory Gerbil Populations in China.

Identifying Helicobacter pylori (H. pylori, Hp) infection in animals before and after artificial infection influences the subsequent experiment. We established effective and noninvasive detection methods, including the gastric fluid nested polymerase chain reaction (PCR) method and the 13C-urea breath test, which can detect Hp before modeling Hp infection in Mongolian gerbils. We designed a gas collection equipment for gerbils. Hp nested PCR was also performed on gastric fluid, gastric mucosa, duodenal contents, and faeces of gerbils challenged with Hp. Conventional Hp detection methods, including rapid urease assay and immunohistochemistry, were compared. Moreover, we assessed the natural infection of Hp in 135 gerbils that had never been exposed to Hp artificially from the major laboratory gerbil groups in China. In 10 Hp infected gerbils, the positive detection results were 100%, 100%, 90%, and 10% in gastric fluid, gastric mucosa, duodenal contents, and faeces with nested PCR, respectively. A rapid urease test performed on gastric mucosa showed that all animals were infected with Hp. Immunohistochemical detection and bacteria culture of gastric mucosa samples that were positive by the nested PCR method also confirmed the presence of Hp. 9% (3/35) and 6% (2/31) natural infection rates were found in conventional gerbil groups from the Capital Medical University and Zhejiang Laboratory Animal Center. In conclusion, we established two noninvasive Hp detection methods that can be performed before modelingHp infection, including the gastric fluid nested PCR method and the 13C-urea breath test.

Development and Assessment of Specific and High Sensitivity Reverse Transcription Nested Polymerase Chain Reaction Method for the Detection of &lt;i&gt;Aichivirus&lt;/i&gt; A Monitoring in Groundwater

Development and Assessment of Specific and High Sensitivity Reverse Transcription Nested Polymerase Chain Reaction Method for the Detection of &lt;i&gt;Aichivirus&lt;/i&gt; A Monitoring in Groundwater

Molecular identification of Pentatrichomonas hominis in animals in central and western Thailand

BackgroundPentatrichomonas hominis inhabits the digestive tracts of several vertebrates, such as humans, monkeys, pigs, dogs, cats and rats. This protozoan was originally considered a commensal of the digestive tract but has subsequently been identified as a potential zoonotic parasite and a causative agent of diarrhoea. Molecular techniques are considered more sensitive and specific to detect P. hominis. This study aimed to determine the presence and genetic diversity of P. hominis in animals in Thailand. A total of 403 faecal samples were collected from 119 cats, 55 dogs, 73 goats, 35 monkeys, 55 cattle and 66 pigs, and the presence of P. hominis was determined using the nested polymerase chain reaction method. Sequence analysis of small-subunit ribosomal RNA genes was used to determine the genotype of the organism.ResultsTwenty-six samples (26/403, 6.45%) were positive for P. hominis. The highest prevalence was found in cats (21/119; 17.65%), followed by cattle (3/55; 5.45%) and dogs (2/55; 3.64%). Seven out of 26 nucleotides demonstrated 100% sequence identity with existing sequences; additionally, 16 novel sequence patterns were identified. All nucleotide sequences of P. hominis-positive samples were shown in the same branch with the previously described P. hominis sequences found in humans, dogs and goat.ConclusionThis is the first study on P. hominis infections in animals in Thailand. Our findings revealed that the prevalence of P. hominis was significantly higher in cats than in cattle and dogs. Cats were the main reservoir host; however, P. hominis can infect several kinds of animals. Therefore, the proper waste management of animals is necessary to reduce and prevent infection in the community.

Association of Human Parvovirus B19 with Papillary Thyroid Carcinoma and its Relationship with Tumor Staging and Pathologic Features

Background: Papillary thyroid carcinoma (PTC) is the most prevalent form of thyroid cancer. In some studies, parvovirus B19 (PVB19) infection was involved in the pathogenesis of thyroid diseases such as Graves’ disease, Hashimoto thyroiditis, and thyroid cancer. PVB19 induces chronic inflammation in thyroid, which can lead to carcinogenesis through the effect of inflammatory mediators. The association of PVB19 with PTC tumorigenesis is still a matter of controversy. We evaluated the correlation of PVB19 with PTC and, for the first time, pathologic features. Method: This cross-sectional retrospective study focused on the thyroid specimens of 82 patients with PTC and 77 patients with benign thyroid nodules. We conducted the present study from March 2014 to November 2017 in the hospitals affiliated with Shiraz University of Medical Sciences, Shiraz, Iran. We evaluated the presence of PVB19 DNA by nested polymerase chain reaction method in PTC, adjacent non-malignant tissues, and benign thyroid nodules. PVB19 positivity was also compared between PTC and two other groups. We further investigated the association of pathologic features and tumor staging with PVB19 positivity. Results: Of the patients, 81% were female. We detected PVB19 positivity in 9.8% of PTC specimens and 0.01 of adjacent non-malignant tissues (P=0.016). None of the benign thyroid nodule specimens had PVB19 DNA, and they were significantly different from PTC specimens (P=0.007). There was no significant correlation between PVB19 positivity and tumor stages (P=0.988) and histologic types (P=0.560). Conclusion: This research, similar to some other studies, showed a significant association between PTC and PVB19 positivity. For the first time, we showed that no significant relationship existed between PVB19 positivity and tumor stages and histologic types. Further investigations are needed to evaluate the relationship between this virus and PTC.

KHV Detection Based on Thermal Lysis - Nested PCR (Polymerase Chain Reaction)

In the case of common carp (Cyprinus carpio) and goldfish (Carrasius auratus) that were lightly infected with Koi Herpes Virus (KHV), the use of the one-step PCR method was insensitive to detect the presence of KHV DNA. The nested PCR method has been developed for a more sensitive detection system for KHV infection. This study aims to test the sensitivity of one-step and nested PCR methods in detecting the KHV virus in lightly infected common carp to prevent early disease attacks. DNA extraction of the test samples used thermal lysis and PBS (Phosphate Buffered Saline). DNA isolate as a KHV template was used to amplify KHV sequences using KHV-TKf: 5'-GGGTTACCTGTACGAG-3 'and KHV-TKr: 5'-CACCCAGTAGATTATGC-3' primers in the one-step PCR method. The nested PCR method uses two pairs of primers, namely the KHV-TKf: 5'-GACACCACATCTGCAAGGAG-3' and KHV-TKr: 5'-GACACATGTTACAATGGTGGC-3') and the CEFAS primer pair (5'-CGTCGTGAGGAATACGACG-3' and 5'- ACCGTACAGCTCGTACTGG-3') for amplification of KHV sequences. The one-step PCR method was insensitive for detection of KHV in common carp and goldfish infected with viruses without clinical symptoms, and was sensitive enough to detect viruses with mild symptoms with a DNA fragment size of 409 bp using TK primers. The nested PCR method was more sensitive in detecting KHV DNA in fish without clinical symptoms with a DNA fragment size of 348 bp using CEFAS primers.

Investigation of Pneumocystis jirovecii Infection and Colonization in Immunocompromised Patients with Pneumonia

Pneumocystis jirovecii is an atypical fungus that causes P.jirovecii pneumonia (PCP) in immunocompromised patients. Currently, while the incidence of AIDS-related PCP is decreasing, PCP has become more common in HIV-negative immunosuppressive patients as a result of increased diseases requiring immunosuppressive therapy. In this study, it was aimed to investigate PCP and colonizations by microscopy, polymerase chain reaction (PCR) and Krebs von den Lungen-6 (KL-6) tests in symptomatic immunosuppressive inpatients with the sign of radiologically atypical pneumonia in Mersin University Hospital. A total of 96 patients, between August 2016 and February 2018 were included in the study. Seventy two (75%) of the 96 patients were under immunosuppressive therapy. P.jirovecii was investigated in the respiratory tract samples [sputum (n= 88), tracheal aspirate (n= 6) and bronchoalveolar lavage (n= 2)] by mtLSUrRNA nested PCR and microscopic staining methods [immunofluorescence assay (IFA), Toluidine Blue O (TBO)], and KL-6 levels were tested in serum samples. P.jirovecii was detected in 16 (16.7%) samples by PCR, in five (5.2%) samples by IFA, in three (3.1%) samples by TBO stain method. When IFA was taken as a reference test, sensitivity and specificity of TBO and PCR were calculated as 60% and 100%; 100% and 87.9%, respectively. In P.jirovecii PCR positive patients, the distribution of underlying diseases; cancer (n= 6), hematological malignancy (n= 3), HIV/AIDS (n= 3), COPD (n= 2), and interstitial lung disease (n= 2) were found as 11 (68.75%) of the 16 positive patients, received immunosuppressive therapy (HIV positive non-Hodgkin lymphoma); of the 3 (18.75%) patients of were immunocompetent, and only 2 (12.5%) were HIV/AIDS. Five of the 16 PCR positive the patients that have positive microscopic examination were definited PCP [HIV/AIDS (n= 3), lung cancer (n= 1), interstitial lung disease (n= 1)]; three patients were PCR positive and microscopy negative probable PCP [multiple myeloma (n= 1), interstitial lung disease (n= 1), cholangiocellular carcinoma (n= 1)] and eight other patients were identified as colonized. In the study, when the frequency of the detection of P.jirovecii was evaluated according to the underlying diseases, it was found statistically significantly higher only in HIV/AIDS patients (p= 0.012). When KL-6 was evaluated among the patients defined as PCP/possible PCP and colonization, sensitivity and specificity were determined as 62.5% and 75%, respectively. As a result, nested PCR method was found as sensitive and successful for the detection of P.jirovecii from sputum samples. KL-6 test was not found sufficient for the differentiation of colonization and the infection in PCR positive patients. The results obtained in the study showed that PCP should be on the differential diagnosis list according to the immune status and the clinical features of the inpatients. More researchs are required with more patients to achieve for detailed reliable results in these groups. In addition, molecular epidemiological studies related to genotyping and resistance against anti-PCP drugs are needed to understand P.jirovecii infections in our region and country.

Development and application of a nested polymerase chain reaction method for the detection of genetically modified soybean in Chinese traditional fermented soy food-sufu

A nested PCR method has been developed to distinguish traditional soybean from transgenic Roundup Ready soybeans (RRS). Comparing with the standard PCR method, the nested PCR method increases the sensitivity by 200 to 1000-fold. One copy of RRS genome can be detected by the nested PCR. Sufu, a traditional fermented soybean curd, is a very important Chinese condiment and it is a highly processed food. We have used the nested PCR to detect seven time points of sufu throughout the aging period. The nested PCR method showed positive results for sufu made up of 100% RRS at all seven time points from 0th day to 180th day. This work describes the development and application of a sensitive and specific detection method for sufu made from RRS using a nested PCR system.

Synthetic oligonucleotide primers to glycoprotein gene of rabies virus and based by nested polymerase chain reaction method for RNA Rabies virus detection

One of widely used methods to detect rabies virus RNA is a reverse transcription chain polymerase reaction (RT-PCR) allowing to determine a diagnosis in 5 hours. Thus, we have elaborated the method that allows to pursue effective elicitation of RNA strains and isolates of rabies virus in pathological and clinical material as well as to decrease research timespan to 6 hours, to lower by 9.8 times the diagnostics prime cost, labor expenditures – by 40 times. The way includes performance of nested RT-PCR with oligonucleotide primers having definite nucleotide sequences and synthesized to conservative glycoprotein gene. Wherein RT-PCR is pursued in two rounds. In the case of positive reaction, fragment corresponding to the size in the first round – 755 bp, in the second round – 259 bp, is synthesized.