Nested PCR Test Research Articles

Gene Polymorphism Of TNF-A (Rs1799964) In Aborted Women With Respiratory Disease In Al-Diwaniyah Province

Background: TNF-α, a proinflammatory cytokine, plays a significant role in the etiology of various illnesses. The encoding gene is found on chromosome 6's short arm in the major histocompatibility complex class III region. Polymorphisms in the TNF-α gene promoter region are believed to impact illness susceptibility and severity. This review summarizes the research on the association between TNF-α gene and receptor polymorphisms and respiratory illness development. This study aim to determine of gene polymorphism of TNF-α in aborted women with respiratory diseases. Methods : The study was carried out for 100 pregnant women, including 60 aborted women with and without respiratory diseases, and 40 women as healthy control group. Genotypes in TNF-α T>C SNPs were identified using self- designed nested T-ARMS PCR tests. Sequencing validated randomly chosen PCR results that represented unique genotypes in TNF-α SNPs. Results : The results of TNF-α (rs1799964) C/T SNP genotyping between aborted women with respiratory diseases and healthy control revealed that the heterozygous C/T genotype was a significant risk factor with an OR of 6.28, while the homozygous TT genotype was non-significant (OR= 2.09). Conclusion : The rs1799964 TNF-α polymorphisms are possible genetic risk factors of respiratory diseases and might be its predictive markers.

Insidensi Ortohantavirus pada Inang Reservoir di Kota Semarang, Jawa Tengah

Orthohantavirus is an RNA virus that causes Hemorrhagic Fever with Renal Syndrome (HFRS) and Hantavirus Pulmonary Syndrome (HPS). Humans can become infected with orthohantaviruses by inhaling virus particles from the urine, saliva, and feces of infective reservoir hosts. Small mammals from the orders Rodensia, Eulipotyphla, and Chiroptera are the main reservoirs of orthohantaviruses. This study aims to detect and calculate the incidence of orthohantavirus in reservoir hosts in Semarang City. The research was conducted descriptively analytically with a cross-sectional study design. The research population was all mice and shrews in the research location (Gayamsari and Tlogosari subdistricts), and the samples were caught mice and shrews (84). The research was carried out in May-September 2023. Orthohantavirus detection was carried out using the Nested PCR test (pre denaturation, denaturation, annealing and extension). A total of 84 individuals were captured, consisting of Rattus novergicus, Rattus tanezumi and Suncus murinus. Orthohantavirus in this study was successfully detected in Rattus norvegicus. The results of the study showed that the incidence of orthohantavirus in Gayamsari Village was (11.1%), Tlogosari Village (2.78%), and the overall total was (2.38%). It is necessary to control mice to prevent transmission of orthohantavirus.

Transmission risk evaluation of transfusion blood containing low-density Babesia microti.

Babesia is a unique apicomplexan parasite that specifically invades and proliferates in red blood cells and can be transmitted via blood transfusion, resulting in transfusion-transmitted babesiosis. However, detecting Babesia in blood before transfusion has not received enough attention, and the risk of transfusing blood containing a low density of Babesia microti (B. microti) is unclear, possibly threatening public health and wellness. This study aimed to determine the lower detection limit of B. microti in blood and to evaluate the transmission risk of blood transfusion containing low-density B. microti. Infected BALB/c mouse models were established by transfusing infected whole blood with different infection rates and densities of B. microti. Microscopic examination, nested Polymerase Chain Reaction (nested PCR), and an enzyme-linked immunosorbent assay (ELISA) were used to evaluate the infection status of the mouse models. Meanwhile, the nested PCR detection limit of B. microti was obtained using pure B. microti DNA samples with serial concentrations and whole blood samples with different densities of B. microti-infected red blood cells. Thereafter, whole mouse blood with a B. microti density lower than that of the nested PCR detection limit and human blood samples infected with B. microti were transfused into healthy mice to assess the transmission risk in mouse models. The infection status of these mice was evaluated through microscopic examination, nested PCR tests, and ELISA. The mice inoculated with different densities of B. microti reached the peak infection rate on different days. Overall, the higher the blood B. microti density was, the earlier the peak infection rate was reached. The levels of specific antibodies against B. microti in the blood of the infected mice increased sharply during the first 30 days of infection, reaching a peak level at 60 days post-infection, and maintaining a high level thereafter. The nested PCR detection limits of B. microti DNA and parasite density were 3 fg and 5.48 parasites/μL, respectively. The whole blood containing an extremely low density of B. microti and human blood samples infected with B. microti could infect mice, confirming the transmission risk of transfusing blood with low-density B. microti. Whole blood containing extremely low density of B. microti poses a high transmission risk when transfused between mice and mice or human and mice, suggesting that Babesia detection should be considered by governments, hospitals, and disease prevention and control centers as a mandatory test before blood donation or transfusion.

Detection of Plasmodium falciparum in Saliva and Stool Samples from Children Living in Franceville, a Highly Endemic Region of Gabon.

Due to the difficulty of obtaining blood samples, which is the invasive method that is currently used for the detection of Plasmodium spp., alternative diagnostic sampling methods that are effective and non-invasive are needed, particularly for long-term studies. Saliva and stool samples from malaria-infected individuals contain trace amounts of Plasmodium DNA and therefore could be used as alternatives. Malaria was screened using rapid diagnosis tests and confirmed via microscopy. Nested PCR tests targeting the Plasmodium falciparum-specific STEVOR gene were performed for blood, saliva and stool samples that were positive for malaria. Three hundred sixty-seven (367) children were enrolled and eighty (22.22%) were confirmed to be positive for malaria. Matched blood, saliva and stool samples were available for 35 children. By using blood smears as the gold standard for the diagnosis of malaria, our study indicates that Plasmodium DNA was more detectable in blood (100%) than in saliva (22.86%) and stools (14.29%). Applying qPCR to the STEVOR gene to detect Plasmodium falciparum DNA in saliva and stool samples cannot be considered as an alternative to the current malaria detection processes using blood specimens.

First report of 'Candidatus Phytoplasma asteris' associated with witches'-broom disease of Pinus yunnanensis in China.

Pinus yunnanensis is an evergreen tree belonging to Pinaceae. The species is distributed in the east of Tibet, southwest of Sichuan, southwest of Yunnan, southwest of Guizhou and northwest of Guangxi. It is an indigenous and pioneer tree species for barren mountain afforestation in southwest China. P. yunnanensis has important value to both the building and medicine industries (Liu et al. 2022). In May 2022, P. yunnanensis showing witches'-broom symptom were found in Panzhihua City, Sichuan Province, China. The symptomatic plants had yellow or red needle, and exhibited plexus bud and needle wither. The lateral buds of infected pines developed into twigs. Some lateral buds grew in clusters and a few sprouted needles (Fig.1). The disease was named the P. yunnanensis witches'-broom disease (PYWB) and was found in some areas of Miyi, Renhe, and Dongqu. More than 9% of the pines showed these symptoms in the three areas surveyed, and the disease was spreading. A total of 39 samples were collected from three areas, including 25 symptomatic plants and 14 asymptomatic plants, respectively. The lateral stem tissues of 18 samples were observed under a scanning electron microscope (Hitachi S-3000N). Spherical bodies were found in the phloem sieve cells of symptomatic pines (Fig.1). Total DNA was extracted from 18 plant samples using the CTAB method (Porebski et al. 1997) and subjected to nested-PCR testing. Double-distilled water and DNA extracted from asymptomatic plants were used as negative controls, and DNA extracted from Dodonaea viscosa affected by the D. viscosa witches'-broom disease was used as positive control. Nested PCR was employed to amplify the pathogen's 16S rRNA gene (Lee et al. 1993; Schneider et al. 1993) and 1.2 kb segment were produced (GenBank accessions OP646619; OP646620; OP646621). PCR specific to the ribosomal protein (rp) gene yielded segment of approximately 1.2 kb (Lee et al. 2003)(GenBank accessions OP649589; OP649590; OP649591). The fragment size from 15 samples was consistent with the positive control, confirming the association of phytoplasma with the disease. A BLAST analysis of the 16S rRNA sequences of P. yunnanensis witches'-broom phytoplasma showed that it shared 99.12% ~99.76% identity with that of Trema laevigata witches'-broom phytoplasma (GenBank accession MG755412). The rp sequence shared 99.84%~99.92% identity with that of Cinnamomum camphora witches'-broom phytoplasma (GenBank accession OP649594). An analysis with iPhyClassifier (Zhao et. 2013) showed that the virtual RFLP pattern derived from OP646621 16S rDNA fragment of PYWB phytoplasma is identical (similarity coefficient 1.00) to the reference pattern of 16Sr group I, subgroup B (OY-M, GenBank accession AP006628). The phytoplasma is identified as a 'Candidatus Phytoplasma asteris'-related strain belonging to sub-group 16SrI-B. Interestingly, compared to AP006628, the virtual RFLP pattern derived from OP646619 and OP646620 fragments exhibit differences in three and one cleavage site, with a similarity coefficient of 0.92 and 0.97, respectively (Fig.2). These strains may represent a new subgroup within the 16Sr group I. The phylogenetic tree was reconstructed based on 16S rRNA and rp gene sequences using MEGA versio6.0 (Tamura et al. 2013). The analysis was conducted using the neighbor-joining (NJ) method with 1,000 replicates of bootstrap analysis. The results indicated that the PYWB phytoplasmas grouped into clades including phytoplasmas belonging to 16SrI-B and rpI-B, respectively (Fig.3). In addition, 2-year-old P. yunnanensis were used for grafting assays in nursery, and the twigs from infected pine under natural conditions were used as a scion, and the phytoplasma were detected using nested PCR after grafting for 40 d (Fig.4). In 2008-2014, P. sylvestris and P. mugo in Lituania had excessive branching symptoms that were attributed to 'Ca. Phtyoplasma Pini' (16SrXXI-A) or asteris' (16SrI-A) strains (Valiunas et al. 2015). In 2015, P. pungens with abnormal shoot branching in Maryland were found to be infected by 'Ca. Phytoplasma pini' strain (16SrXXI-B) (Costanzo et al. 2016). To the best of our knowledge, P. yunnanensis is a new host of 'Ca. Phytoplasma asteris'-related strain (16SrI-B) in China. The newly emerged disease is a threat to pines.

PET-PCR reveals low parasitaemia and submicroscopic malarial infections in Honduran Moskitia

BackgroundMalaria remains a main parasitic disease of humans. Although the largest number of cases is reported in the African region, there are still endemic foci in the Americas. Central America reported 36,000 malaria cases in 2020, which represents 5.5% of cases in the Americas and 0.015% of cases globally. Most malaria infections in Central America are reported in La Moskitia, shared by Honduras and Nicaragua. In the Honduran Moskitia, less than 800 cases were registered in 2020, considering it an area of low endemicity. In low endemicity settings, the number of submicroscopic and asymptomatic infections tends to increase, leaving many cases undetected and untreated. These reservoirs challenge national malaria elimination programmes. This study aimed to assess the diagnostic performance of Light Microscopy (LM), a nested PCR test and a photoinduced electron transfer polymerase chain reaction (PET-PCR) in a population of febrile patients from La Moskitia.MethodsA total of 309 febrile participants were recruited using a passive surveillance approach at the Puerto Lempira hospital. Blood samples were analysed by LM, nested PCR, and PET-PCR. Diagnostic performance including sensitivity, specificity, negative and positive predictive values, kappa index, accuracy, and ROC analysis was evaluated. The parasitaemia of the positive samples was quantified by both LM and PET-PCR.ResultsThe overall prevalence of malaria was 19.1% by LM, 27.8% by nPCR, and 31.1% by PET-PCR. The sensitivity of LM was 67.4% compared to nPCR, and the sensitivity of LM and nPCR was 59.6% and 80.8%, respectively, compared to PET-PCR. LM showed a kappa index of 0.67, with a moderate level of agreement. Forty positive cases by PET-PCR were not detected by LM.ConclusionsThis study demonstrated that LM is unable to detect parasitaemia at low levels and that there is a high degree of submicroscopic infections in the Honduran Moskitia.

Deteksi Plasmodium knowlesi Menggunakan Nested Polymerase Chain Reaction (PCR) di Kecamatan Muara Komam Kalimantan Timur

Plasmodium knowlesi is a parasite of the genus plasmodium that naturally infects long-tailed macaques (Macaca fascicularis), but currently reported has ability to infect humans. The identification/detection of P. knowlesi can be done using RDT, microscopic, or molecular examinations using nested PCR. Nested PCR is the most sensitive and specific method of examination to date. This study aimed to detect P. knowlesi in humans by RDT, microscopic, and nested PCR examinations. The study was descriptive with a cross-sectional approach, carried out from March to July 2019. The samples in this study were 123 patients who were suspected of being infected with malaria and who underwent laboratory tests at the Muara Komam Health Center. Microscopic examination and RDT examination were carried out at the Muara Komam Health Center, while nested PCR was carried out at the Eijkman Molecular Biology Laboratory Jakarta. The results of RDT and microscopic examinations showed as many as 16 of 123 (13%) malaria-positive samples of P. falciparum and P. vivax, and 10 of 123 (8.1%) malaria-positive samples of P. falciparum and P. vivax. Nested PCR tests targeting the rRNA SSU gene were able to identify P. knowlesi by 6 out of 123 (4.87%). In conclusion, the study showed that Plasmodium knowlesi was detected in humans in Muara Komam, East Kalimantan through nested PCR examination.

The Investigation of Giardiasis (Foodborne and Waterborne Diseases) in Buffaloes in Van Region, Türkiye: First Molecular Report of Giardia duodenalis Assemblage B from Buffaloes

Giardia duodenalis (G. duodenalis) is an important zoonotic protozoan agent that causes foodborne and waterborne diarrhea in humans and other mammals. Molecular-based tests are critical in diagnosing giardiasis in humans and animals, identifying species, understanding the zoonotic potential and transmission routes, and evaluating taxonomy. Therefore, this study aimed to investigate the molecular characterization of G. duodenalis in buffaloes in the Van region in Türkiye. Buffaloes are a species that has been poorly studied in this regard. For this purpose, 100 fecal samples were collected from buffaloes in the Van region. The DNA extraction was performed using the GeneMATRIX STOOL DNA Purification Kit from stool samples. The nested PCR test was performed with the appropriate primers from the obtained DNA samples. The obtained bands suitable for sequencing were sent for sequence analysis, and the sequence results were aligned bidirectionally and compared with the database of GenBank by BLAST. As a result of the study, an 11% positivity rate for G. duodenalis was found in buffaloes, and assemblage E and assemblage B were isolated. To our knowledge, assemblage B in buffaloes was reported for the first time in this study. As a result, it was concluded that buffaloes are an important reservoir for waterborne and foodborne giardiasis.

The Pathology of Fatal Avian Malaria Due to Plasmodium elongatum (GRW6) and Plasmodium matutinum (LINN1) Infection in New Zealand Kiwi (Apteryx spp.).

Avian malaria caused by Plasmodium species is a known cause of mortality in avifauna worldwide, however reports within New Zealand kiwi (Apteryx spp.) are scant. Postmortem reports from kiwi were obtained from the Massey University/Te Kunenga ki Pūrehuroa School of Veterinary Science Pathology Register from August 2010-August 2020. Gross lesions were described from postmortem reports, and archived H.E.-stained slides used for histological assessment. Nested PCR testing was performed on formalin-fixed paraffin-embedded tissue samples to assess the presence of Plasmodium spp. and Toxoplasma gondii DNA and cases with a PCR-positive result were sequenced to determine the lineage involved. Of 1005 postmortem reports, 23 cases of confirmed or suspected avian malaria were included in this study. The most consistent gross lesions included splenomegaly, hepatomegaly, and interstitial pneumonia with oedema. Histological lesions were characterised by severe interstitial pneumonia, pulmonary oedema, interstitial myocarditis, hepatic sinusoidal congestion and hypercellularity, and splenic macrophage hyperplasia and hyperaemia/congestion with numerous haemosiderophages. Cytoplasmic meronts were consistently found within endothelial cells of a variety of tissues, and within tissue macrophages of the liver, lung and spleen. A diagnosis of avian malaria was confirmed via PCR testing in 13 cases, with sequencing revealing P. matutinum (LINN1) and P. elongatum (GRW6) as the species involved. This is the largest case series describing the pathology of avian malaria as a cause of mortality in endemic New Zealand avifauna.

Emerging Tick-Borne Pathogens in Central Canada: Recent Detections of Babesia odocoilei and Rickettsia rickettsii.

Background: The spread of emerging tick-borne pathogens has steadily increased in Canada with the widespread establishment of tick vectors and vertebrate hosts. At present, Borrelia burgdorferi, the bacterium causing Lyme disease, is the most common tick-borne pathogen in Canada and primarily transmitted by Ixodes scapularis. A low prevalence of other emerging tick-borne pathogens, such as Anaplasma phagocytophilum, Babesia species, Borrelia miyamotoi, and Francisella tularensis have also been detected through surveillance efforts in Canada. Although Rickettsia rickettsii has been historically detected in Haemaphysalis leporispalustris in Canada, the current prevalence and geographic extent of this pathogen is unknown. Material and Methods: In this study, we assessed the presence and prevalence of several emerging tick-borne pathogens in ticks and hosts collected through tick dragging and small mammal trapping in Central Canada. Results: Nested PCR testing detected three pathogen species in ticks, with Babesia odocoilei and B. burgdorferi in I. scapularis in addition to R. rickettsii in H. leporispalustris. Three pathogen species were detected in small mammals by nested PCR including B. odocoilei in Blarina brevicauda, Babesia microti in Peromyscus leucopus, and a Hepatozoon species in P. leucopus and Peromyscus maniculatus. B. burgdorferi and Babesia species were the pathogens most often detected in our samples, suggesting they are widely distributed across Central Canada. We also detected B. odocoilei and R. rickettsii beyond their known geographic distribution. Conclusions: Our results provide evidence that emerging tick-borne pathogens may be present outside defined risk areas identified by current surveillance efforts in Canada. As a result, emerging tick-borne pathogens introduced by the dispersal of infected ticks by migratory birds or maintained by hosts and vectors through cryptic transmission cycles may go undetected. More comprehensive testing including all tick life stages and additional tick-borne pathogens will help detect the spread and potential risk of emerging or re-emerging tick-borne pathogens for human and wildlife populations throughout Canada.

Türkiye’nin Iğdır İli’nde sararma ve çalımsı görünüm gösteren biber bitkilerinde ‘Candidatus phytoplasma trifolii’nin varlığı

Objective: The objective of this study was to investigate the group/subgroup of phytoplasma agent in peppers showing phytoplasma symptoms. Material and Methods: In this study, plants collected from Iğdır province in 2020 were analyzed using direct and nested PCR tests, and BLASTn, iphyClassifier, Mega 7, and pDRAW32 programs were used. Results: In the tests performed, approximately 1.2 kb of DNA fragments specific to phytoplasma were obtained. The 16S rRNA nucleotide sequence (1254 bp in length) (OM663745) revealed that it was showed more than 99.44% nucleotide similarity to other ‘Ca. P. trifolii’ members. The tentative RFLP and phylogenetic analyzes performed proved the ‘Ca. P. trifolii’ the infection from the Clover proliferation group (16SrVI) group and subgroup A in symptomatic pepper plants. Conclusion: The presence of ‘Ca. P. trifolii’ in naturally infected peppers in Iğdır province of Turkey was detected using PCR-RFLP and cladistic analysis.

Extraction of the DNA of Giardia lamblia isolated from vegetables and fruits in a simplified way and its diagnosis using Nested-PCR.

Giardiasis is one of the main diseases transmitted by food contaminated with infectious stages. It was usually spread through freshly eaten food. In this study, this parasite was diagnosed based on microscopic diagnosis of isolated samples of vegetables and fruits, as well as molecular diagnosis. The possibility of isolating parasites from vegetables and fruits and extract DNA to be used in most laboratories were verified in this study .The prevalence of Giardia lamblia were estimated on vegetables and fruits without the need to purchase a special extraction kit. .The prevalence of Giardia on fresh vegetables was estimated by using wet direct swab with Lugol's iodine dye in microscopic examination, at a rate of 4 positive samples out of 230 of all studied species, or 1.7%. DNA extraction was carried out for all samples, and 102 DNA samples were extracted from all samples, with a percentage of 44.3%. Moreover, the shown results of the nested PCR test for the 16S rRNA gene on the obtained DNA the prevalence of Giardia lamblia at a rate of 5.9% from DNA samples isolated from vegetables and fruits.

Comparative Detection of Canine Parvovirus by Differential PCR, Conventional PCR and Nested PCR tests: Detecting Antigenic Variants by Differential PCR

Canine parvovirus (CPV) is a pathogen causing hemorrhagic enteritis in puppies and mainly transmitting via feco-oral route. In this study, stool samples were collected from a total of 35 animals suspected of CPV. The samples were examined by Conventional PCR, Nested PCR and Differential PCR tests. 20 out of 35 dogs (57.1%) were detected positive by conventional PCR, 31 (88.6%) by nested PCR and 30 (85.7%) by differential PCR. CPV 2a was stated as the most common antigenic type, male animals and 0–3-month-olds had a high rate of becoming sick and vaccinated animals might also catch the disease, rarely. Accordingly, it is recommended to focus on studies providing molecular epidemiology surveillance in order to detect the existing subtypes and develop reliable diagnosis and vaccination methods.

Occurrence of Echinococcus spp. in red foxes and wolves in the protected area of the Tatra National Park in southern Poland - a threat to human health.

Echinococcus multilocularis has been endemic in red foxes in eastern and central parts of Europe, and E.granulosus s. l. identified in wolves in some countries. In recent years, wolves hale emerged as potentially important definitive hosts of E. multilocularis. This aim of the survey was to record indirectly using nested-PCR test with faecal samples the presence of Echinococcus multilocularis and E. granulosus s. l. in the two species of wild canids in the protected area of the Tatra National Park (TNP) in Western Carpathian, southern mountainous part of Poland. From February to June 2019, experienced staff of TNP randomly collected fox and wolf faeces on and off hiking trails at altitudes from 850 m to 2,000 m above sea level. In total, 91 faecal samples from red foxes and 19 from wolves were collected. Genomic DNA was obtained by direct extraction from faecal samples using a commercial kit, and from taeniid eggs retrieved from the same samples after flotation. A nested PCR screening of 91 red fox faeces indicated the prevalence of E. multilocularis of 4.4%. Positive samples were confirmed by sequencing parts of the mitochondrial cytochrome c oxidase subunit 1 gene (cox1). Neither genomic DNA of E. multilocularis nor of E. granulosus s.l. was obtained from 19 wolves faeces, nor from taeniid eggs retrieved from these samples by initial flotation. The current results show that humans might be exposed to a risk of fox tapeworm infection in nature, even at high altitude inan alpine zone, in an environment contaminated by roaming red foxes encouraged by food leftovers on mountain trails.

Impact of Varicella-Zoster Virus/ Hepatitis C Virus (VZV/HCV) co-infection on liver enzymes activity

Varicella is a common, benign and self-limiting disease. It is related to hepatitis and malfunction/failure of the liver. This study was designed to assess Varicella-Zoster Virus/ Hepatitis C Virus (VZV/HCV) co-infection on the levels of liver enzymes. A total of 88 blood samples regardless of Hepatitis C Virus positivity were collected. The age and sex of the sample population are included in the demographic statistics. HCV-RNA was detected by RTnested PCR and VZV-DNA was identified by nested PCR in all the sera. Approximately 27 patients were positive for HCV and VZV co-infection. Liver function was also tested on the basis of parameters such as Aspartate Amino Transferase (AST) and Alanine Amino Transferase (ALT). Infection with HCV and VZV is more prevalent in the 34-55 age group tested by nested-PCR and liver function tests based on Alt and AST levels using qualitative test kits. The ALT and AST levels were high 102 and 99.6 IU/L in co-infected cases as compared to 35 and 32 IU/L in normal cases without HCV, HIV and VZV. The study results indicate the impact of VZV in the abnormality of liver function. This study concludes that coinfection with HCV/VZV can be seen as a highrisk category for the development of liver disease where long-term outcomes of the disease should be controlled.

Investigation of soil contaminated with Toxoplasma gondii oocyst in urban public environment, in Brazil

In this study, we determined the occurrence of Toxoplasma gondii oocysts in soil samples from public places. A total of 120 samples were collected from 24 sites, including squares, parks, university, hospitals in the city of Recife. The recovered oocysts were subjected to a nested-PCR test, and nine sites (9/24) were found to be positive for gene of apicomplexan parasites. The PCR product was sequenced, and 8.33% (10/120) of the samples showed 100% similarity to T. gondii DNA. T. gondii oocysts were detected in 75% (3/4) of the evaluated hospital soil samples and in 23.81% (5/21) soils samples from the public squares and parks. The results of this study demonstrate the potential of the soil in the areas analyzed as a source of T. gondii infection and therefore highlight the importance of devising educational strategies on the use of these sites, in addition to future cleaning protocols in public areas.

Detection of Cacao Mild Mosaic Virus (CaMMV) Using Nested PCR and Evidence of Uneven Distribution in Leaf Tissue

Distribution of improved germplasm of Theobroma cacao is essential for meeting the increased demand for cocoa beans. In cacao, the introduction of new diseases is prevented by exchanging material through a national and international quarantine system. In 2020, virus symptoms were observed on plants in a quarantine greenhouse, and Cacao mild mosaic virus (CaMMV) was detected in one plant using published diagnostic primers. However, no virus was detected in other symptomatic plants. To address high pathogen diversity and low virus titer in recently infected plants, a nested PCR test was developed based on 15 CaMMV sequences from Trinidad and Puerto Rico. The test was validated on a subset (n = 30) of plants in the greenhouse, of which 29 tested positive. Most infections are thought to have occurred during the later stage of the quarantine period, possibly due to spread by mealybugs. However, phylogenetic analysis revealed the presence of three strains, suggesting that it was introduced on scionwood from multiple sources. Results of PCR assays on different leaf tissues indicate that the virus is unevenly distributed and that petiole tissue should be used in molecular diagnostics. The movement of infected scionwood is a major dissemination pathway for CaMMV but can be managed through careful screening.

Detection of Brucella Abortus Using Nested-PCR Molecular Method

Common detection methods of brucellosis are always accompanied by technical problems in infection diagnosis. In this study, the nested PCR technique was used for the detection of Brucella abortus, and it was compared with other methods. This case-control study was done in Kurdistan. Blood samples were collected from 50 patients with clinical symptoms and 50 healthy people with no clinical symptoms. Serologic and culture tests were done for all the samples. DNA was extracted using a Promega kit. Molecular detection was done by nested PCR. In the end, the results of phenotypical method and nested PCR were compared. The results of culture and serology tests indicate the limited capabilities of these methods to detect Brucella species. According to the results among all samples, only 14 (28%) were cultured. Rose Bengal test sensitivity was 100% but its specificity 26.09%. Also, Wright's test (tube agglutination) sensitivity was also 85% and nested PCR sensitivity was 46.15%. The sensitivity of Rose Bengal, Wright's and nested PCR tests were 100%, 85%, and 46.15%, respectively, for this sampling.

Analysis of initial laboratory diagnosis of malaria and its accuracy compared with re-testing from 2013 to 2018 in Yunnan Province, China

BackgroundAccording to China’s Malaria Eradication Action Plan, malaria cases diagnosed and reported by health authorities at the county level must be further re-confirmed by provincial laboratories. The Yunnan Province Malaria Diagnostic Reference Laboratory (YPMDRL) began the synchronous implementation of microscopic examinations and nested polymerase chain reaction (nested-PCR) testing to re-test the malaria cases initially diagnosed by county-level laboratories and to evaluate the consistency of Plasmodium species identified between by YPMDRL and by the county-level laboratories from 2013 to 2018 in Yunnan Province.MethodsData on malaria initial diagnosis completed by county-level laboratories in Yunnan Province were collected weekly from the “China Disease Prevention and Control Information System” from 2013 to 2018. The YPMDRL performed Plasmodium microscopic examination and 18S rRNA gene nested-PCR testing on every malaria case managed by the China Disease Prevention and Control Information System. The re-testing detection results were fed back to the initial diagnosis and reporting unit for revision of malaria case types.ResultsA total of 2,869 malaria cases were diagnosed and reported by county-level laboratories in Yunnan Province from 2013 to 2018. The re-testing rate was 95.6% (2,742/2,869), and the re-testing rate increased from 2013 to 2018. Among the re-tested 2,742 cases, 96.7% (2651/2742), 2.2% (59/2742), and 1.1% (32/2742) were doubly examined by microscopy and by nested-PCR, only by microscopy, and only by nested-PCR, respectively. The total Plasmodium species accuracy rate at county-level laboratories was 92.6% (2,543/2,742) reference to the diagnosis by YPMDRL. Among the inconsistent 199 cases, they were identified as including 103 negative cases, 45 falciparum malaria cases, 30 vivax malaria cases, 11 ovale malaria cases, and 10 malariae malaria cases by YPMDRL. From 2013 to 2018, the revised and registered malaria cases by the China Disease Prevention and Control Information System in Yunnan Province was 2,747 cases, including 2,305 vivax malaria cases, 421 falciparum malaria cases, 11 ovale malaria cases, and 10 malariae malaria cases.ConclusionsThe double re-testing strategy by microscopy and by gene testing increases the accuracy of diagnoses malaria in Yunnan Province, and gene testing can reliably differentiate Plasmodium species. The re-testing results provided by YPMDRL are the authoritative basis for revising malaria kind in Yunnan Province.

Stolbur and Clover Proliferation Phytoplasma Infections in Tomato from Bingöl province, Turkey

Phytoplasma-type symptoms were noted in tomato (Lycopersicum esculentum L.) in Bingöl province of Turkey. The remarkable symptoms include witch’s broom, rosetting, purple curled and crispy leaves, small leaves in the upper branches and excessively elongated calyx. Genomic DNA isolation was performed to identify possible pathogens from the leaves of 11 plants with and without symptom. In the Nested-PCR test performed using universal primer sets, DNA bands of approximately 1200 bp size were obtained in 4 of 11 samples. Randomly chosen two DNA bands primed R16F2n/R16R2 were cloned into an appropriate plasmid vector to further characterizations. The recombinant plasmid DNAs purified were sequenced in both directions. Molecular assays of the 16S rRNA sequence confirmed the existence of the “Canditatus Phytoplasma solani” (16SrXII-A group) (similarity coefficient 1.00) (Accession no: MT279680) and the “Canditatus Phytoplasma trifolii” (16SrVI-A group) (similarity coefficient 1.00) (Accession no: MT279852) in the infected tomato samples. The isolates associated with tomato-phytoplasma were named as ‘Bingöl D11’ and ‘Bingöl D90’ isolates, respectively. The phylogenetic dendrogram created also confirmed where both pathogens belong. This current paper is documented in the first record of “Ca. P. solani” (16SrXII-A) and “Ca. P. trifolii” (16SrVI-A) in naturally diseased tomato in Bingol of Turkey.