White spot syndrome virus (WSSV) transmission risk through infected cooked shrimp products assessed by polymerase chain reaction (PCR) and bio-inoculation studies

The aim of the study was to evaluate the resistance of white spot syndrome virus (WSSV) in shrimps (Penaeus monodon) to the process of cooking. The cooking was carried out at 1000C six different durations 5, 10, 15, 20, 25 and 30 min. The presence of WSSV was tested by single step and nested polymerase chain reaction (PCR). In the single step PCR, the primers 1s5 & 1a16 and IK1 & IK2 were used. While in the nested PCR, primers IK1 &IK2 – IK3 & IK4 were used for the detection of WSSV. WSSV was detected in the single step PCR with the primers 1s5 and 1a16 and the nested PCR with the primers IK1 and IK2 – IK3 & IK4 from the cooked shrimp samples. The cooked shrimps, which gave positive results for WSSV by PCR, were further confirmed for the viability of WSSV by conducting the bio-inoculation studies. Mortality (100%) was observed within 123 h of intra-muscular post injection (P.I) into the live healthy WSSV-free shrimps (P. monodon). These results show that the WSSV survive the cooking process and even infected cooked shrimp products may pose a transmission risk for WSSV to the native shrimp farming systems

Semi-nested PCR para a detecção molecular de Paracoccidioides brasiliensis em amostras de tecido

INTRODUCTION: Paracoccidioidomycosis is a systemic infection caused by Paracoccidioides brasiliensis. METHODS: In this study, a semi-nested PCR for paracoccidioidomycosis diagnosis was developed. The primers ITS1 and ITS4 were used in the first reaction, while the primers MJ03 and ITS1 primer were used in the second reaction. The semi-nested PCR was used to investigate biopsies of five patients with oral lesions that resembled paracoccidioidomycosis. RESULTS: The semi-nested PCR was positive for four samples and negative for a sample from a patient later diagnosed with leishmaniasis. CONCLUSIONS: The new semi-nested PCR describe is useful for aracoccidioidomycosis diagnosis. ________________________________________________________________________________________________________________ RESUMOINTRODUÇÃO: A paracoccidioidomicose é uma infecção sistêmica causada pelo Paracoccidioides brasiliensis. MÉTODOS: Neste estudo, uma semi-nested PCR foi desenvolvida para o diagnóstico da paracoccidioidomicose. Os oligonucleotídeos iniciadores ITS1 e ITS4 foram usados na primeira reação, enquanto os oligonucleotídeos iniciadores MJ03 e ITS1 foram usados na segunda reação. A semi-nested PCR foi usada para investigar biopsias de cinco pacientes com lesões orais que se assemelhavam a paracoccidioidomicose. RESULTADOS: A semi-nested PCR foi positiva para quatro amostras e negativa para a amostra de um paciente, posteriormente diagnosticado com leishmaniose. CONCLUSÕES: A semi-nested PCR descrita aqui é útil para o diagnóstico da paracoccidioidomicose

Human Infections with Plasmodium knowlesi, the Philippines.

Five human cases of infection with the simian malaria parasite Plasmodium knowlesi from Palawan, the Philippines, were confirmed by nested PCR. This study suggests that this zoonotic infection is found across a relatively wide area in Palawan and documents autochthonous cases in the country

Toxoplasma Gondii Identification in Mother\u27s Blood and Fetal Tissue with Nested PCR

Toxoplasma gondii identification in mother’s blood and fetal tissue with nested PCRObjective: To examine the correlation between Toxoplasma gondii infection with spontaneous abortion on pregnant women based on nested PCR result from mother’s blood and fetal tissue.Methods: A prospective clinical diagnostic study using nested PCR performed on 30 cases of pregnant women with spontaneous abortion fulfilling the inclusion criteria, latex agglutination test (+) and exclusion criteria, latex agglutination test (-). Mother’s blood and fetal tissues samples, which gave positive result in serologic test, were analyzed with nested PCR using18S-rDNA gene primers.Results: Five of 30 mother’s blood samples (16.7%) and 9 of 30 fetal tissue samples (30%) gave positive PCR results. According to Fisher’s Exact test, PCR detected the presence of Toxoplasma gondii in significant value (P < .001). Conclusion: There is a strong correlation between Toxoplasma gondii infection with spontaneous abortion (P < 001)

Molecular Detection of Anaplasma bovis in Cattle from Central Part of Iran

Anaplasma bovis is a leukocytotropic agent of bovine anaplasmosis and there is no available information about molecular study on this agent in cattle of Iran. In this study a total 150 cattle blood samples were collected from central part of Iran. The presence of A. bovis examined using light microscopic detection and species-specific nested polymerase chain reaction (nested-PCR) based on 16S rRNA gene. Of the 150 cattle, 4 (2.66 %) was positive for A. bovis by nested-PCR. These data is the first A. bovis DNA presence in cattle from central part of Iran

Development of an Rt Nested PCR-Elisa Diagnostic Test for the Detection of Newcastle Disease Virus

Newcastle disease virus causes an economically important poultry disease known as Newcastle disease in Malaysia. The velogenic strain of this virus causes 1 00% mortality in infected chicken. Therefore, a rapid and sensitive diagnostic method is necessary for the detection of the virus. In this study a sensitive one tube reverse transcription nested PCR was developed by using two nested pairs of primer which were designed from the consensus fusion gene sequences. The outer primer sequences are 5'-T ACACCTCATCCCAGACAGGGTC-3' (FOP1) and 5'- AGGCAGGGGAAGTGATTTGTGGC-3' (FOP2). The inner primer pair with the sequence of 5'-T ACTTTGCTCACCCCCCTT-3' (FIP1) and 5'-CATCTTCCCAACTGCCACT-3' (FIP2) were labeled with biotin and digoxigenin at their 5' ends respectively. The PCR condition for the outer primers is 90°C/30 s, 67°C/30 s and 72°C/30 s for 20 cycles in which a 532 bp PCR product was generated. While for the inner primers, the PCR condition used is 90°C/30 s, 55°C/30 s and 72°C/15 s for a total of 30 cycles for the amplification of a labeled 280 bp PCR product. The primer pairs used are highly specific enabling the identification of all the three different pathotypes of NDV. No cross-reactions with other avian infectious agents such as infectious bronchitis virus, infectious bursal disease virus, influenza virus, and fowl pox virus were observed. The detection limit of this one tube nested peR technique was 3 pfu/ml of NDV by agarose gel electrophoresis detection method and was about 100 fold more sensitive compared to that of a non-nested RT - peR. To facilitate the detection of the peR product, the amplified peR product was then subjected to a colorimetric detection method using ELIS A where the labeled peR product was captured in a streptavidin coated microtiter plate and was detected by using anti-digoxigenin-peroxidase enzyme conjugate and 2,2'-azinodiethylbenzothiazolinesulfonic acid (ABTS) as the substrate. Comparisons between the detection methods of agarose gel electrophoresis and ELISA showed that the latter was 10-fold more sensitive than the former. The efficacy of the nested PCR-ELIS A was also compared with the conventional NDV detection method (HA test) and nonnested RT-PCR by testing against a total of 35 tissue specimens collected from NDsymptomatic chickens. With the cutoff value of 0.154 having been calculated from 15 known negative samples, 21 of 35 (60%) samples were tested to be NDV positive by nested peR-ELISA. One of these positive samples, however, was negative by nested peR and gel detection method. Only 8 of 35 (22.9%) samples were tested positive by non-nested RT-PCR and 2 of 35 (5.7%) samples were positive by the conventional HA test. Due to the high sensitivity of nested PCR-ELISA for the detection of NDV from tissue specimens, a peR-ELISA based diagnostic test may be a useful screening test especially in dealing with large number of samples

Cryptosporidium infection in patients with gastroenteritis in Sari, Iran

Background: Cryptosporidiosis is a common coccidian parasite infection in patients with diarrhea that has worldwide distribution especially in developed countries. Therefore, the aim of this study was to determine the occurrence of Cryptosporidium infection in patients with gastroenteritis admitted to hospitals of Mazandaran University of Medical Sciences by parasitological and molecular methods in Sari, Iran. Methods: Stool samples were collected from 348 patients with gastroenteritis admitted to the hospitals of Medical University in the Sari and Ghaemshahr cities in Mazandaran Province, Northern Iran in 2010-2011. Oocysts of Cryptosporidium identified using Formalin-Ether concentration method and stained by Aacidfast staining (AFS) and Auramine phenol fluorescence (APF). Genomic DAN extracted from microscopically positive samples and nested PCR -RFLP by using SSU rRNA that identifies of the species of cryptosporidium. Results: In 348 patients with gastroenteritis, the most clinical symptoms were diarrhea, nausea, vomiting, dehydration, fever and weight loss. 2.3% (8 cases) of diarrheal samples tested by both microscopy and molecular methods were positive for the presence of cryptosporidium. Nested PCR products yielded unique bands of 846 bp, correspond to cryptosporidium. Species diagnosis carried out by digesting the secondary PCR product with SspI restriction enzyme, which noted 3 clearly bands of 449, 254, and 108 bp correspond to Cryptosporidium spp. Conclusion: The results of present study on Cryptosporidium spp. in this area can make a background data for control programs and further molecular analyses. Thus, further work needs to determine the origin of Cryptosporidium species in this area

Phytoplasma detection in coconut palm and other tropical crops

Phytoplasmas are small bacteria with very small genomes which also have extremely low levels of the nucleotides guanine plus cytosine (G+C). They are associated with hundreds of plant diseases globally. The uneven distribution and low concentration of phytoplasmas in the phloem of infected plant, especially in woody hosts, and variations in titre according to season and plant organ are also important obstacles for efficient diagnosis. Polymerase Chain Reaction (PCR), nested PCR and real-time PCR have been employed for phytoplasma detection. PCR is the most versatile tool for detecting phytoplasmas in their plant and insect hosts. Nested PCR with a combination of different universal primers can improve the diagnosis of unknown phytoplasmas present with low titre in the symptomatic host. Universal ribosomal primers nested with group-specific primers are extremely useful when the phytoplasma to be diagnosed belongs to a well-defined taxonomic group. Real-time PCR has been shown to be an effective method of quantifying the titre of phytoplasmas within the plant. This paper also discuss phytoplasma diseases on coconut palm