Direct Diagnosis of Trichomonas vaginalis Infection on Archived Pap Smears Using Nested PCR

2015 ◽  
Vol 59 (1) ◽  
pp. 104-108 ◽  
Author(s):  
Hajar Ziaei Hezarjaribi ◽  
Mahbobeh Taghavi ◽  
Mahdi Fakhar ◽  
Shirzad Gholami
Keyword(s):  
Dna Extraction ◽  
Study Design ◽  
Nested Pcr ◽  
Trichomonas Vaginalis ◽  
Direct Detection ◽  
Urogenital Tract ◽  
Pap Smears ◽  
Direct Pcr ◽  
Parasitic Protozoan ◽  
Archived Samples

Objectives: Little information is available concerning PCR-based direct detection of Trichomonas infections on archived Pap (Papanicolaou)-stained smears. This study investigates DNA extraction and amplification from archived Pap smears. Trichomonas vaginalis is a parasitic protozoan that infects the urogenital tract of women. Study Design: DNA from archived Pap-stained smears was successfully amplified using the nested PCR to investigate if it could be used for accurate detection and retrospective epidemiological investigations. Results: In our study, 98 (75.4%) out of 130 specimens of T. vaginalis Pap-stained smears were found to be positive by the nested PCR. Also, direct PCR on the archived Pap smears for identifying T. vaginalis gave a specificity of 100%. Conclusion: PCR-based Pap smears appear to offer an effective method to detect Trichomonas infection in archived samples, being rapid, highly specific and convenient for sampling, particularly in retrospective investigations.

scholarly journals Toxoplasma gondii in Sheep and Goats from Central Iran

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Mojtaba Bahreh ◽  
Bahador Hajimohammadi ◽  
Gilda Eslami
Keyword(s):  
Toxoplasma Gondii ◽  
Dna Extraction ◽  
Infection Rate ◽  
Nested Pcr ◽  
Prevention Programs ◽  
Central Iran ◽  
Infection Rates ◽  
Sheep And Goats ◽  
One Year ◽  
Undercooked Meat

Abstract Objective Toxoplasmosis, caused by Toxoplasma gondii, infects humans by consuming infected raw or undercooked meat and foods harboring mature oocysts. In this study, we assessed the prevalence of T. gondii in sheep and goats coming from central Iran. After completing the questionnaire, about one gram of liver or diaphragm tissue was taken as a sample from 90 sheep and 90 goats slaughtered in Yazd Province and stored at – 20 ºC. DNA extraction was done, and then T. gondii was detected using nested PCR. Results This study indicated that the prevalence of T. gondii in all slaughtered animals was 11.6% (21 of 180), including 14.4% (13/90) in sheep and 8.8% (8/90) in goats. The infection rates in liver and diaphragm samples were 12.2% (11/90) and 11.1% (10/90), respectively (p = 0.8163). The infection rate in animals older than one was 16.3% (15/92), and it was 6.8% (6/88) in animals under one year of age. Therefore, no significant differences were found (p = 0.475). Infection rates were 19.5% (18/92) in males and 3.4% (3/88) in females (p = 0.0007). In conclusion, the infection rates of toxoplasmosis in livestock in this area are almost high, and therefore, it is necessary to design appropriate prevention programs to control the disease.

Development of a rapid DNA extraction method and one-step nested PCR for the detection of Naegleria fowleri from the environment

2011 ◽  
Vol 45 (16) ◽  
pp. 5211-5217 ◽  
Author(s):  
Arine Fadzlun Ahmad ◽  
James Lonnen ◽  
Peter W. Andrew ◽  
Simon Kilvington
Keyword(s):  
Dna Extraction ◽  
Nested Pcr ◽  
Extraction Method ◽  
Naegleria Fowleri ◽  
Dna Extraction Method ◽  
One Step ◽  
Nægleria Fowleri

scholarly journals Direct PCR from whole blood, without DNA extraction

1990 ◽  
Vol 18 (19) ◽  
pp. 5908-5909 ◽  
Author(s):  
B. Mercier ◽  
C. Gaucher ◽  
O. Feugeas ◽  
C. Mazurier
Keyword(s):  
Dna Extraction ◽  
Whole Blood ◽  
Direct Pcr

scholarly journals Detection of Bacteria in Environmental Samples by Direct PCR Without DNA Extraction

BioTechniques ◽  
2001 ◽  
Vol 31 (3) ◽  
pp. 598-607 ◽  
Author(s):  
Kimberly A. Fode-Vaughan ◽  
Charles F. Wimpee ◽  
Charles C. Remsen ◽  
Mary Lynne Perille Collins
Keyword(s):  
Dna Extraction ◽  
Environmental Samples ◽  
Direct Pcr

scholarly journals First Report of Flavescence Dorée-Related Phytoplasma Affecting Grapevines in Croatia

Plant Disease ◽  
2011 ◽  
Vol 95 (3) ◽  
pp. 353-353 ◽  
Author(s):  
M. Šeruga Musić ◽  
D. Škorić ◽  
I. Haluška ◽  
I. Križanac ◽  
J. Plavec ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Reference Strain ◽  
Simultaneous Detection ◽  
Pinot Noir ◽  
Vitis Vinifera L ◽  
Rrna Gene ◽  
Pcr Assay ◽  
Direct Pcr ◽  
Flavescence Dorée ◽  
Scaphoideus Titanus

Flavescence dorée (FD) and Bois noir (BN) phytoplasmas are principal grapevine yellows (GY) agents in the wider Euro-Mediterranean Region. While BN phytoplasma belongs to the ribosomal subgroup 16SrXII-A, the FD agents belong either to the ribosomal subgroups 16SrV-C or -D. During the official GY survey in 2009, 40 symptomatic grapevines (Vitis vinifera L.) were sampled throughout grapevine-growing regions in Croatia. Typical GY symptoms of leaf yellowing or reddening were evident on white and red varieties, respectively. Leaf rolling as well as irregular lignification of the shoots and withering of clusters were also observed. Phloem tissue from cuttings and leaf veins from mature vines were sampled for total DNA extraction and amplification of phytoplasma 16S rRNA gene by using generic primers P1/P7 in a direct PCR assay followed by a nested PCR using primer pair R16F2n/R2 (2). Phytoplasma ribosomal group affiliation was determined by restriction fragment length polymorphism (RFLP) analysis of the nested PCR products with enzyme Tru1I (Fermentas, Vilnius, Lithuania). These initial findings were validated and augmented by a triplex real-time PCR assay targeting the nonribosomal map gene. This assay enables simultaneous detection of BN and FD (16SrV-C and -D) phytoplasmas in grapevine (3). Assay results revealed the majority of GY positive vines (19 of 40) contained BN phytoplasma which is widespread. For the first time in Croatia, two red variety samples, Pinot Noir and Plemenka Crvena, from the vicinity of Ozalj (Vivodina) and Zagreb (Brezje), respectively, were found to harbor FD-related phytoplasmas. Fragments amplified by P1/P7 primers from latter samples were cloned and sequenced. Sequence analyses using online interactive tool iPhyClassifier (4) revealed that the phytoplasma under study from Pinot Noir sample (GenBank Accession No. HQ712064) is a member of 16SrV-C subgroup and shares 99.87% similarity with 16S rDNA sequence of the reference strain (GenBank Accession No. AF176319). The sequence from the Plemenka Crvena sample (GenBank Accession No. HQ712065) shares 99.54% similarity with the reference strain and has the most similar virtual RFLP pattern to the one of the 16SrV-C subgroup (GenBank Accession No. AY197642). These findings are currently limited to vineyards in northwestern Croatia. Even so, the presence of FD principal cicadellid vector Scaphoideus titanus in the country and the occurrence and distribution of FD in neighboring countries (1,2) are factors indicating that the spread of FD in Croatia is highly probable. References: (1) L. Filippin et al. Plant Pathol. 58:826, 2009. (2) S. Kuzmanović et al. Vitis 47:105, 2008. (3) C. Pelletier et al. Vitis 48:87, 2009. (4) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

scholarly journals First Report of a Group 16SrII Phytoplasma Associated with Witches'-Broom of Eggplant in Oman

Plant Disease ◽  
2011 ◽  
Vol 95 (3) ◽  
pp. 360-360 ◽  
Author(s):  
A. M. Al-Subhi ◽  
N. A. Al-Saady ◽  
A. J. Khan ◽  
M. L. Deadman
Keyword(s):  
Nested Pcr ◽  
Solanum Melongena ◽  
Positive Control ◽  
Rrna Gene ◽  
Direct Pcr ◽  
First Report ◽  
Pcr Product ◽  
Pcr Products ◽  
Broom Disease ◽  
Phytoplasma Infection

Eggplant (Solanum melongena L.) belongs to the family Solanaceae and is an important vegetable cash crop grown in most parts of Oman. In February 2010, plants showing phyllody symptoms and proliferation of shoots resembling those caused by phytoplasma infection were observed at Khasab, 500 km north of Muscat. Total genomic DNA was extracted from healthy and two symptomatic plants with a modified (CTAB) buffer method (2) and analyzed by direct and nested PCR with universal phytoplasma 16S rDNA primers P1/P7 and R16F2n/ R16R2, respectively. PCR amplifications from all infected plants yielded an expected product of 1.8 kb with P1/P7 primers and a 1.2-kb fragment with nested PCR, while no products were evident with DNA from healthy plants. Restriction fragment length polymorphism (RFLP) profiles of the 1.2-kb nested PCR products of two eggplant phyllody phytoplasma and five phytoplasma control strains belonging to different groups used as positive control were generated with the restriction endonucleases RsaI, AluI, Tru9I, T-HB8I, and HpaII. The eggplant phytoplasma DNA yielded patterns similar to alfalfa witches'-broom phytoplasma (GenBank Accession No. AF438413) belonging to subgroup 16SrII-D, which has been recorded in Oman (1). The DNA sequence of the 1.8-kb direct PCR product was deposited in GenBank (Accession No. HQ423156). Sequence homology results using BLAST revealed that the eggplant phyllody phytoplasma shared >99% sequence identity with Scaevola witches'-broom phytoplasma (Accession No. AB257291.1), eggplant phyllody phytoplasma (Accession No. FN257482.1), and alfalfa witches'-broom phytoplasma (Accession No. AY169323). The RFLP and BLAST results of 16S rRNA gene sequences confirm that eggplant phyllody phytoplasma is similar to the alfalfa phytoplasma belonging to subgroup 16SrII-D. To our knowledge, this is the first report of a phytoplasma of the 16SrII-D group causing witches'-broom disease on eggplant in Oman. References: (1) A. J. Khan et al. Phytopathology 92:1038, 2002. (2) M. A. Saghai-Maroof et al. Proc. Natl. Acad. Sci. USA, 81:8014, 1984.

scholarly journals Direct detection of microorganisms responsible for the sexually transmitted infection on the mobile real-time PCR device without treating in advance

2021 ◽  
Author(s):  
Masaaki Muraoka ◽  
Kazunori Sohma ◽  
Osamu Kawaguchi ◽  
Mikio Mizukoshi
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Direct Detection ◽  
Treponema Pallidum ◽  
Nucleic Acid Amplification ◽  
Direct Pcr ◽  
Transmitted Infection ◽  
Sexually Transmitted ◽  
Ct Value

ABSTRACTAs WHO reported, four curable STIs-chlamydia, gonorrhoea, syphilis and trichomoniasis occur more than 1 million per each day globally almond 2016. For this reason, it is important to control these STIs, one of which is “to detect”. The general methods in order to detect STIs are nucleic acid amplification tests (NAATs). One of the reasons why NAATs are utilized in many tests is that it is possibly to be more sensitive than other test. However, there needs to treat extraction of nucleic acids in advance and amplify specific regions by NAATs, and hence it must take much labour and much time. In this work, for Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and Treponema pallidum (TP) which is each etiological agent of chlamydia, gonorrhoea and syphilis, we evaluate and propose “quicker and simpler” NAATs. Specifically, utilizing mobile real-time PCR device “PCR1100” and PCR reagent kit “KAPA3G Plant PCR Kit”, it was considered whether real-time direct PCR could be performed or not without treating DNA extraction in advance so-called “direct”.As a result, firstly, we established that real-time direct PCR could be performed in all of CT, NG, and TP, and moreover, each Ct value correlated with the concentration of each organism similarly to detection of genome DNA (each correlation coefficient R2 > 0.95). Moreover, each assay demonstrated a limit of detection (LOD) of the follows; CT was 10^0.86 = 7.24 IFU/reaction, NG was 10^-0.19 = 0.65 CFU/reaction, and TP was 10^1.4 = 25.1 organisms/reaction. However, it appeared the sensitivity was a little low, especially for CT and TP.Secondly, we found that even as without treating sample in advance, the time of detection was required more less 15 minutes at any of case, which was very quick compared with other current methods for real-time PCR. Additionally, compared with other commercial devices, it was easier to operate the PCR1100 device, for example, start, analysis of Ct value.In conclusion, the present study has demonstrated that it is possible for real-time direct PCR to perform with combination of the PCR1100 device and the PCR reagent kit in 3 kinds of microorganisms-CT, NG and TP. Furthermore, we propose “quicker and simpler” methods for NAATs, which it would not take labour and time. Further studies are needed in order to contribute to control STIs.

scholarly journals Direct Identification of Vibrio vulnificus in Clinical Specimens by Nested PCR

1998 ◽  
Vol 36 (10) ◽  
pp. 2887-2892 ◽  
Author(s):  
Shee Eun Lee ◽  
Soo Young Kim ◽  
Sei Jong Kim ◽  
Hyun Soo Kim ◽  
Jong Hee Shin ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Nested Pcr ◽  
Extraction Method ◽  
Vibrio Vulnificus ◽  
High Yield ◽  
Chromosomal Dna ◽  
Direct Identification ◽  
Clinical Specimens ◽  
Dna Extraction Method ◽  
Dna Fragment

This study was performed to establish optimal nested PCR conditions and a high-yield DNA extraction method for the direct identification ofVibrio vulnificus in clinical specimens. We designed two sets of primers targeting the V. vulnificushemolysin/cytolysin gene. The target of the first primer set (P1-P2; sense, 5′-GAC-TAT-CGC-ATC-AAC-AAC-CG-3′, and antisense, 5′-AGG-TAG-CGA-GTA-TTA-CTG-CC-3′, respectively) is a 704-bp DNA fragment. The second set (P3-P4; sense, 5′-GCT-ATT-TCA-CCG-CCG-CTC-AC-3′, and antisense, 5′-CCG-CAG-AGC-CGT-AAA-CCG-AA-3′, respectively) amplifies an internal 222-bp DNA fragment. We developed a direct DNA extraction method that involved boiling the specimen pellet in a 1 mM EDTA–0.5% Triton X-100 solution. The new DNA extraction method was more sensitive and reproducible than other conventional methods. The DNA extraction method guaranteed sensitivity as well, even when V. vulnificus cells were mixed with other bacteria such asEscherichia coli or Staphylococcus aureus. The nested PCR method could detect as little as 1 fg of chromosomal DNA and single CFU of V. vulnificus. We applied the nested PCR protocol to a total of 39 serum specimens and bulla aspirates from septicemic patients. Seventeen (94.4%) of the 18V. vulnificus culture-positive specimens were positive by the nested PCR. Eight (42.1%) of the 19 culture-negative samples gave positive nested PCR results.

scholarly journals Clinical and microbial characterization of toxigenic Clostridium difficile isolated from antibiotic associated diarrhea in Egypt

2020 ◽  
Author(s):  
Sherein G. Elgendy ◽  
Sherine A. Aly ◽  
Rawhia Fathy ◽  
Enas A.E. Deaf ◽  
Naglaa H. Abu Faddan ◽  
...  
Keyword(s):  
Risk Factors ◽  
Clostridium Difficile ◽  
Pediatric Patients ◽  
Direct Detection ◽  
Diagnostic Methods ◽  
Adult Patients ◽  
Specific Method ◽  
Healthcare Associated Infection ◽  
Direct Pcr ◽  
Antibiotic Associated Diarrhea

Background and Objectives: Clostridium difficile infection (CDI) has become a significant healthcare-associated infection throughout the world and is particularly important in developing countries. This study aimed to investigate clinical characterization and risk factors related to toxigenic C. difficile infection in adult and pediatric patients, antimicrobial susceptibility pattern. Also, to evaluate different diagnostic methods for rapid detection of C. difficile associated diarrhea (CDAD) in Egypt. Materials and Methods: Stool samples were collected from 95 pediatric patients and 37 adult patients suffering from antibiotic associated diarrhea and were subjected to direct toxin immunoassay and culture on cycloserine/cefoxitin/fructose agar. The presence of tcdA and tcdB genes was tested by PCR. Results: Toxigenic C. difficile was isolated from pediatric and adult patients at a rate of 17.89% (17/95) and 27% (10/37) respectively. The sensitivity and specificity of direct PCR from stool are (100%, 100% and 82.4%, 100%) in adult and pediatric samples respectively. The susceptibility of C. difficile to vancomycin and metronidazole were found to be 66.7% and 48.2% respectively. Conclusion: Diabetes mellitus, prior antibiotic treatment, hematological malignancy on chemotherapy, malnutrition, neutropenia and Ryle feeding are risk factors for development of CDAD. Tight restriction of unnecessary antibiotic uses is necessary in our locality. Direct detection of toxin genes in stool by PCR is sensitive and specific method for early detection of C. difficile.

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