scholarly journals Diagnosis of Blastocystis sp., Cryptosporidium sp., and Giardia intestinalis by Multiplex PCR: An Optimization Study

2021 ◽  
Author(s):  
Ali Ahmet Kilimcioğlu ◽  
Nogay Girginkardeşler ◽  
Tuba Oyur ◽  
Selin Bölük Sabuncu ◽  
Didem Düzyol Azak ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Dna Isolation ◽  
Giardia Intestinalis ◽  
Molecular Method ◽  
Chain Reaction ◽  
Optimization Study ◽  
Optimized Protocol ◽  
Blastocystis Sp ◽  
Stool Samples ◽  
Polymerase Chain

Objective: It was aimed to develop a new Multiplex Polymerase Chain Reaction (PCR) protocol with isolates obtained from local patients for the diagnosis of Blastocystis sp., Cryptosporidium sp. and Giardia intestinalis, which can cause severe gastrointestinal system complaints especially in immunocompromised patients and children. Method: DNA isolation was performed with a commercial kit from three stool samples of different patients whose microscopic examination showed dense amounts of Blastocystis sp., Cryptosporidium sp. and Giardia intestinalis. First, a special PCR protocol has been developed for each protozoon. Then, the multiplex PCR protocol, in which these three protozoa can be diagnosed together, was optimized. Results: In the multiplex PCR protocol performed after DNA isolation, bands of 95 bp., 227 bp. and 258 bp. were obtained for Cryptosporidium sp., Blastocystis sp. and G. intestinalis, respectively. Conclusion: Blastocystis sp., Cryptosporidium sp. and Giardia intestinalis were diagnosed by multiplex PCR with the original protocol developed. Due to the difficulties in using different methods in parasitological examination, by adding other protozoa important for public health to this optimized protocol, it will be possible to detect a large number of parasites with a single molecular method.

scholarly journals Prevalence and Subtype Distribution of Blastocystis sp. in Senegalese School Children

2020 ◽  
Vol 8 (9) ◽  
pp. 1408
Author(s):  
Salma Khaled ◽  
Nausicaa Gantois ◽  
Amadou Tidjani Ly ◽  
Simon Senghor ◽  
Gaël Even ◽  
...  
Keyword(s):  
School Children ◽  
Epidemiological Survey ◽  
Single Infection ◽  
Chain Reaction ◽  
Blastocystis Sp ◽  
Northwestern Region ◽  
Waterborne Transmission ◽  
Stool Samples ◽  
Polymerase Chain ◽  
Enteric Protozoan

Blastocystis sp. is an enteric protozoan that frequently colonizes humans and many animals. Despite impacting on human health, data on the prevalence and subtype (ST) distribution of Blastocystis sp. remain sparse in Africa. Accordingly, we performed the first multicenter and largest epidemiological survey ever conducted on Blastocystis sp. for this continent. A total of 731 stool samples collected from healthy school children living in 10 villages of the northwestern region of Senegal were tested for the presence of Blastocystis sp. by real-time polymerase chain reaction followed by subtyping of positive samples. Considerable variation in prevalence between villages (51.7 to 100%) was evident with the overall prevalence being 80.4%. Mixed infections were identified in 23% of positive individuals. Among 453 school children with a single infection, ST2 was predominant, followed by ST1, ST3, ST7, ST10, and ST14; this is the first report of ST10 and ST14 in humans. Genetic polymorphisms were evident at the intra-ST level with the identification of numerous ST1 to ST3 genotypes. ST1 showed the greatest intra-ST diversity followed by ST2 and ST3. The prevalence and distribution of STs and genotypes varied among target villages, pointing to several potential infection sources, including human-to-human, zoonotic, and waterborne transmission.

scholarly journals OPTIMAL CONDITION FOR MULTIPLEX POLYMERASE CHAIN REACTION (PCR) IN DETECTING ASCARIS LUMBRICOIDES, TRICHURIS TRICHIURA, AND NECATOR AMERICANUS IN PRESERVED STOOL

2020 ◽  
Vol 5 (1) ◽  
pp. 34
Author(s):  
Christiane Marlene Sooai ◽  
Elsa Herdiana M ◽  
Supargiyono Supargiyono
Keyword(s):  
Multiplex Pcr ◽  
Dna Isolation ◽  
Coi Gene ◽  
Chain Reaction ◽  
Microscopic Method ◽  
Optimal Method ◽  
Complex Process ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Dna Template

Background: Multiplex PCR examination is one of the molecular examination methodologies applied to detect soil-transmitted helminth (STH) infection. Optimization of the multiplex PCR method is a complex process, but it is necessary to obtain both a correct detection process and a satisfactory DNA product. Objective: To determine whether multiplex PCR can be optimized to diagnose STH from Indonesian isolates, and to find the optimal method for detection of A. lumbricoides, T. trichiura, and N. americanus infections in stool that have been stored for 3 years. Methods: A total of 15 samples were examined, and these samples were previously examined by using a microscopic method, then continued with optimization steps. Result: The optimal PCR mixture used primers targeting COI gene for A. lumbricoides, 18S rDNA for T. trichiura and ITS1 for N. americanus, 15 µl of Go Taq Green Master Mix, 5 µl of the 3 pairs of primers, 5 µl of DNA template and 4 µl of DdH2O, and the condition was 30 minutes of 950C denaturation, 30 second of 530C annealing and 1 minute of 720C extension, repeated for 35 cycles. Conclusion: Multiplex PCR can be optimized for STH detection from Indonesian isolates. The successful detection using the multiplex PCR method was influenced by sample preparation prior to DNA isolation, which includes several steps i.e. homogenization of samples using bead beaters and passing samples on liquid nitrogen rapidly.

scholarly journals Seasonal Differences in Cyclospora cayetanensis Prevalence in Colombian Indigenous People

2021 ◽  
Vol 9 (3) ◽  
pp. 627
Author(s):  
Hagen Frickmann ◽  
Juliane Alker ◽  
Jessica Hansen ◽  
Juan Carlos Dib ◽  
Andrés Aristizabal ◽  
...  
Keyword(s):  
Indigenous People ◽  
Rainy Season ◽  
Gastrointestinal Symptoms ◽  
Dry Season ◽  
Seasonal Effects ◽  
Chain Reaction ◽  
Resource Limited Settings ◽  
Resource Limited ◽  
Stool Samples ◽  
Polymerase Chain

Fecal-orally transmitted cyclosporiasis is frequent in remote resource-limited settings in Central and South America with poor hygiene conditions. In this study, we aimed at assessing seasonal effects on the epidemiology of colonization or infection with C. cayetanensis in Colombian indigenous people living under very restricted conditions. In the rainy season between July and November and in the dry season between January and April, stool samples from indigenous people with and without gastrointestinal symptoms were collected and screened for C. cayetanensis applying in-house real-time polymerase chain reaction (PCR). In the rainy season and in the dry season, positive PCR results were observed for 11.8% (16/136) and 5.1% (15/292), respectively, with cycle threshold (Ct) values of 30.6 (±3.4) and 34.4 (±1.6), respectively. Despite higher parasite loads in the rainy season, fewer individuals (2/16, 12.5%) reported gastrointestinal symptoms compared to the dry season (6/15, 40%). In conclusion, considerable prevalence of C. cayetanensis in Colombian indigenous people persists in the dry season. Low proportions of gastrointestinal symptoms along with higher parasite loads make colonization likely rather than infection.

Evaluation of polymerase chain reaction and DNA isolation protocols for detection of genetically modified soybean

2007 ◽  
Vol 42 (10) ◽  
pp. 1249-1255 ◽  
Author(s):  
Cibele dos Santos Ferrari ◽  
Luciana Lehmkuhl Valente ◽  
Fábio Cristiano Angonesi Brod ◽  
Caroline Tagliari ◽  
Ernani Sebastião Sant'Anna ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Isolation ◽  
Genetically Modified ◽  
Chain Reaction ◽  
Genetically Modified Soybean ◽  
Polymerase Chain

DNA Isolation from Recalcitrant Materials Such as Tree Roots, Bark, and Forest Soil for the Detection of Fungal Pathogens by Polymerase Chain Reaction

1998 ◽  
Vol 262 (1) ◽  
pp. 79-82 ◽  
Author(s):  
Günther Bahnweg ◽  
Steffen Schulze ◽  
Evelyn M. Möller ◽  
Hilkea Rosenbrock ◽  
Christian Langebartels ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Forest Soil ◽  
Fungal Pathogens ◽  
Dna Isolation ◽  
Chain Reaction ◽  
Tree Roots ◽  
Polymerase Chain

Diagnostic Value of Multiplex Polymerase Chain Reaction in Detection of Acinetobacter baumannii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia from Sepsis in Pediatrics

2021 ◽  
Vol 15 ◽  
Author(s):  
Sara Galeb ◽  
Maysaa El Sayed Zaki ◽  
Raghdaa Shrief ◽  
Rasha Hassan ◽  
Mohamed Anies
Keyword(s):  
Polymerase Chain Reaction ◽  
Pseudomonas Aeruginosa ◽  
Acinetobacter Baumannii ◽  
Multiplex Pcr ◽  
Stenotrophomonas Maltophilia ◽  
Multiplex Polymerase Chain Reaction ◽  
Diagnostic Value ◽  
Blood Cultures ◽  
Chain Reaction ◽  
Polymerase Chain

Background: Proper identification of the causative organism in pediatric sepsis is crucial for early diagnosis and prevention of septic shock and organ failure. Objectives: The present study aimed to evaluate the multiplex Polymerase Chain Reaction (PCR) to detect Acinetobacter baumannii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia from positive blood cultures for these pathogens isolated from children, with hospital-acquired sepsis compared to the conventional biochemical reactions for identification of these organisms. Methods: This study was a cross-sectional study performed on 100 isolates from pediatric blood cultures, including Acinetobacter baumannii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia. The study also included 100 isolates of Escherichia coli as a negative control. All isolates were identified by API 20NE and the multiplex PCR, with primers specific to the 3 tested bacteria. Results: Multiplex PCR was positive in 96% of isolates, and 4 isolates had negative results. False positive results were reported with three E. coli strains. Multiplex PCR identified all the isolates of Acinetobacter baumannii, 29 isolates of Pseudomonas aeruginosa, and 27 isolates of Stenotrophomonas maltophilia. Compared to the biochemical identification, the diagnostic value of the multiplex PCR revealed 96.04% sensitivity, 96.9% specificity, 97.00%, positive predictive value, 96.00% negative predictive value, and 96.50% accuracy. Conclusion: The present study highlights the diagnostic value of multiplex PCR to identify Acinetobacter baumannii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia from positive blood cultures. Multiplex PCR was sensitive, specific, and accurate. The accuracy differs according to the organisms, with 100% accuracy for Acinetobacter baumannii.

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