scholarly journals Penggunaan Empat Set Primer dalam Mendeteksi Tryapanosoma Evansi pada Mencit dengan Teknik Polymerase Chain Reaction (PCR)

2019 ◽  
Vol 1 (1) ◽  
pp. 13-19
Author(s):  
Dias Aprita Dewi ◽  
Fitrine Ekawasti ◽  
April H. Wardhana ◽  
Dyah H. Sawitri
Keyword(s):  
Polymerase Chain Reaction ◽  
Sensitivity And Specificity ◽  
Filter Paper ◽  
High Sensitivity ◽  
Culture Collection ◽  
Male Mice ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Primer Sets

Diagnose technique for detection Tryapanosoma evansi have been developed for many years, including Polymerase Chain Reaction (PCR). One of fundamental factors on this technique is primer desain which possess high sensitivity and specificity. The aim of the current study was to examine four primer sets for detection of T. evansi on mice. They were ITS 1, ESAG 6/7, RoTat 1.2 VSG, TBR1/2 and TR3/TR4. Balitvet Culture Collection (BCC) – Bangkalan isolate of T. evansi was was injected into two male mice DDY strain. Three days after infection, blood mice was collected and stored at -20oC. In addition, some blood was dropped on the filter paper and kept at 4oC. Another mice was killed and some tissues (brain, liver, lung, heart, spleen and kidney) were removed from mice and preserved into 80% Ethanol and then they were stored -20oC. All samples were amplified using five primer sets and repeated twice. The results demonstrated that all primers (ITS 1, ESAG 6/7, RoTat 1.2 VSG, TBR1/2 and TR3/TR4) were able to detect T. evansi on all samples (the blood, the filter paper and all tissues). In order to detect T. evansi in the field, primer ITS-1 is recommended because it is able to discriminate some spesies T. evansi in livestock and generate a good intensity of DNA band from tested samples.

scholarly journals POLYMERASE CHAIN REACTION AS A DIAGNOSTIC TOOL FOR SIX SEXUALLY TRANSMITTED INFECTIONS - PRELIMINARY RESULTS

2015 ◽  
Vol 88 (1) ◽  
pp. 33-37
Author(s):  
Alecsandra Iulia Grad ◽  
Mihaela Laura Vica ◽  
Horea Vladi Matei ◽  
Doru Lucian Grad ◽  
Ioan Coman ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Chlamydia Trachomatis ◽  
Neisseria Gonorrhoeae ◽  
Sexually Transmitted Infections ◽  
Sensitivity And Specificity ◽  
Diagnostic Tool ◽  
High Sensitivity ◽  
Chain Reaction ◽  
Sexually Transmitted ◽  
Polymerase Chain

Background and aim. Sexually transmitted infections are a very frequent and under-diagnosed cause of illness worldwide. A high number of detection methods and a large range of specimens in which sexually transmitted infections can be determined are available at the moment. Polymerase chain reaction performed on first void urine offers the advantage of being non-invasive, self-collectable and has high sensitivity and specificity. We looked to determine the frequency of Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, Mycoplasma hominis, Mycoplasma genitalium and Ureaplasma urealyticum in symptomatic and asymptomatic patients.Methods. Six sexually transmitted infections were determined in the first void urine of 15 symptomatic and asymptomatic patients by polymerase chain reaction. We used “Epicenter MasterPure™ Complete DNA and RNA Purification Kit” for the DNA purification and “Seeplex® STD6 ACE Detection” for the DNA amplification. The results were examined in UV light.Results. A number of 5 patients had positive results for Chlamydia trachomatis or Neisseria gonorrhoeae. Sexually transmitted infections are more frequent in men between 27 and 40 years old.Conclusions. Polymerase chain reaction is a good diagnostic tool for sexually transmitted infections because it has a high sensitivity and specificity. Chlamydia trachomatis is the most frequent sexually transmitted infection, followed by Neisseria gonorrhoeae.

scholarly journals LOOP-MEDIATED ISOTHERMAL AMPLIFICATION FOR DETECTION OF C. BURNETII IN BULGARIA

2021 ◽  
Vol 19 (2) ◽  
pp. 147-151
Author(s):  
M. Kunchev ◽  
V. Belcheva ◽  
E. Grigorov
Keyword(s):  
Polymerase Chain Reaction ◽  
Sensitivity And Specificity ◽  
Q Fever ◽  
High Sensitivity ◽  
Isothermal Amplification ◽  
Chain Reaction ◽  
Retrospective Diagnosis ◽  
Loop Mediated Isothermal Amplification ◽  
The World ◽  
Polymerase Chain

Q fever, which is caused by Coxiella burnetii, a small, pleomorphic intracellular bacterium, is the most widespread zoonosis in the world. The chronic form of the disease can lead to disability and death. Rapid diagnosis of Q fever is needed in order that effective treatment can be initiated. The conventional retrospective diagnosis of Q fever, based on serology, is useless for the treatment of afflicted patients. Thus, molecular methods have been created to close the diagnostic gap between the onset of the disease and the presence of specific antibodies in serum. A polymerase chain reaction is a suitable and reliable method with high sensitivity and specificity, but it requires expensive equipment and post-amplification protocol. Loop-mediated isothermal amplification (LAMP) is an isothermal technique, conducted at constant temperature that can amplify a negligible amount of DNA to more than 109 copies within one hour, using special primers and polymerase. We have tested the sensitivity and specificity of LAMP in the detection of C. burnetii. The mean positive rate of LAMP and polymerase chain reaction in patients was 100% and 74%, respectively. LAMP reacted negatively with non-C. burnetii pathogens and non-infected blood samples. We conclude that LAMP is a sensitive and specific technique for the detection of C. burnetii and has advantages over serological methods and PCR that make it attractive for diagnosing Q fever in countries around the world.

scholarly journals Polymerase chain reaction chlamydia trachomatis examination in nonspecific genital infection patients

2019 ◽  
Author(s):  
Dian Pertiwi Habibie ◽  
Dwi Murtiastutik ◽  
R. Rahmadewi
Keyword(s):  
Polymerase Chain Reaction ◽  
Chlamydia Trachomatis ◽  
Sensitivity And Specificity ◽  
High Sensitivity ◽  
Genital Infection ◽  
Chain Reaction ◽  
Causal Organism ◽  
Advanced Method ◽  
Polymerase Chain ◽  
Low Incidence

Introduction: Nonspecific genital infection (NSGI) is an inflammation of urethra, rectum, or cervix that caused by nonspecific bacteria. Chlamydia trachomatis is known as the most causal organism of NSGI, usually mild (mucopurulent discharge) or asymptomatic, and if untreated it can cause serious complication such as pelvic inflammatory disease that leads to infertility in women. The diagnosis of Chlamydia trachomatis needs an advanced method, such as polymerase chain reaction (PCR). PCR has high sensitivity and specificity, and endocervical swab is specimen of choice that also has high sensitivity and specificity to diagnose Chlamydia trachomatis. Method: This research aims to evaluate if Chlamydia trachomatis is the most causal organism of NSGI by PCR Chlamydia trachomatis using 201bp primers. Results: Eighteen NSGI married patients who came to outpatient clinic were evaluated from endocervical swab. The result demonstrated that 16,67% from eighteen NSGI patient positive Chlamydia trachomatis. Conclusion: The low incidence of Chlamydia trachomatis in low risk population such in this study need further study, the cause of NSGI needs to be known certainly so the exact treatment can be given.

scholarly journals PERBANDINGAN HASIL DETEKSI PLASMODIUM SPP ANTARA CARA PEMERIKSAAN MIKROSKOPIK TETESAN DARAH TEBAL DAN TEKNIK POLYMERASE CHAIN REACTION

2014 ◽  
Vol 6 (1) ◽  
Author(s):  
Cindy Sandra ◽  
Josef S. B. Tuda ◽  
Victor D. Pijoh
Keyword(s):  
Polymerase Chain Reaction ◽  
Diagnostic Test ◽  
Sensitivity And Specificity ◽  
Predictive Value ◽  
Blood Smear ◽  
High Sensitivity ◽  
Thick Blood Smear ◽  
Chain Reaction ◽  
Genus Plasmodium ◽  
Polymerase Chain

Abstract: Malaria is still a health problem in the world, especially in undeveloped countries. This disease is caused by protozoa of the genus Plasmodium and has two ways of transmission, naturally (Anopheles spp.) and unnaturally. WHO mentioned that in 2006 there were as many as 200-300 million cases of clinical malaria with 880,000 deaths. In 2012, it was recorded that there were a total of 8,691 malaria cases in North Sulawesi, Indonesia. Therefore, an early diagnostic tool with high sensitivity and specificity is needed. This study aimed to compare the sensitivity and specificity in detection of Plasmodium spp by using thick blood smear method to Polymerase Chain Reaction (PCR). This was a diagnostic test using blood samples of 30 malaria patients at Budi Mulia Hospital and Manembo-nembo Hospital Bitung from September 2013 - January 2014. Thick blood smears were prepared and microcopically tested, then the specimens were scrapped and be further tested by using the PCR. The microscopic test showed 20 positive samples meanwhile the PCR showed 24 positive samples. A diagnostic test using predictive table 2x2 indicated that the PCR had 100% sensitivity in general, 60% specifity, 83.3% positive predictive value, and 100% negative predictive value. Conclusion: Compared to the thick blood smear, the PCR was more accurate in detecting plasmodia in malaria cases with a moderate specificity value and a high sensitivity value.Keywords: thick blood smear, Polymerase Chain Reaction (PCR), sensitivity, specificity  Abstrak: Malaria merupakan salah satu penyakit yang masih menjadi masalah kesehatan di dunia terutama di negara-negara yang belum berkembang. Malaria disebabkan oleh protozoa dari genus Plasmodium dan ditularkan melalui dua cara yaitu alamiah melalui nyamuk Anopheles spp. dan tidak alamiah. WHO melaporkan bahwa pada 2006 terdapat 200-300 juta kasus malaria dengan 880.000 kematian. Di Provinsi Sulawesi Utara, Indonesia, dilaporkan total kasus malaria tahun 2012 sebesar 8.691. Oleh karena itu diperlukan suatu alat diagnostik dini dengan sensitivitas dan spesifisitas yang baik. Penelitian ini bertujuan untuk membandingkan tingkat sensitivitas dan spesifisitas hasil deteksi Plasmodium spp antara cara pemeriksaan mikroskopik tetesan darah tebal dan teknik Polymerase Chain Reaction (PCR). Penelitian ini merupakan uji diagnostik dengan sampel darah dari 30 pasien malaria di RS Budi Mulia dan RS Manembo-nembo Bitung sejak September 2013 - Januari 2014. Setelah disiapkan tetesan darah tebal, dilakukan pemeriksaan mikroskopik. Selanjutnya, spesimen darah dikerok dan diperiksa dengan PCR. Hasil pemeriksaan mikroskopik menunjukkan 20 sampel positif malaria sedangkan pemeriksaan PCR 24 sampel positif malaria. Tes uji diagnostik dengan tabel prediktif 2x2 mendapatkan tingkat sensitivitas PCR secara umum sebesar 100%, spesifisitas 60%, nilai duga positif 83,33%, dan nilai duga negatif 100%. Simpulan: Dibandingkan tetesan darah tebal, pemeriksaan PCR dapat mendeteksi secara lebih akurat adanya plasmodia pada kasus malaria, dengan nilai spesifitas sedang dan nilai sensitivitas tinggi.Kata kunci: tetesan darah tebal, Polymerase Chain Reaction (PCR), sensitivitas, spesifisitas

scholarly journals Genotyping of Listeria by Polymerase Chain Reaction (PCR) and its Epidemiological Significance

2021 ◽  
Vol 6 (2) ◽  
pp. 115-124
Author(s):  
V. Zyuzin ◽  
◽  
O. Tuzova ◽  
U. Frenkel ◽  
Muntian L. ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Internal Control ◽  
Pcr Analysis ◽  
Chain Reaction ◽  
Virulence Markers ◽  
Chip Technology ◽  
Polymerase Chain ◽  
Primer Sets ◽  
Epidemiological Significance

The purpose of the study. The article covers the issues of genotyping of listeria by polymerase chain reaction (PCR) and its epidemiological significance. It is known that molecular genetic methods allow to detect specific microbial pathogens, virulence markers, antimicrobial resistance genes faster and with greater sensitivity than traditional culture methods. Therefore, the development of detection methods and genotyping by polymerase chain reaction (PCR) is relevant. Materials and methods. For the detection and genotyping of Listeria, the technology of DNA chips is becoming increasingly important, which can significantly expand the possibilities of molecular detection. Chip technology can be used to simultaneously identify a whole range of pathogenic microorganisms, to determine genetic virulence markers, the relationship to antibiotics, subtyping, as well as to determine the quality of microorganisms in samples. A simplified version of DNA chip technology is multiplex (numerical) PCR, which is used to detect and genotype listeria. Studies have shown that to detect Listeria spp. using a polymerase chain reaction, it is advisable to use the gene iap (invasive associated protein), known for 6 species of listeria, which encodes a protein P 60 that is common to all species of listeria, including L.murrayi. Computer analysis revealed areas with 100% homology, from which primers were selected for PCR detection of all types of listeria. Areas of genomes characterized by 100% homology were selected for further analysis and labeling of primer sets. The sequences of the constructed primers List 1 and List 2 allowed to identify 6 species of Listeria (L. monocytogenes, L. innocua, L. ivanovii, L. grayi, L. seeligeri, L. welshimeri). Increasing the length of the primer leads to the increasing of specificity of PCR analysis. The greater the length of the primer, the smaller the specific gravity of one error of the unpaired nucleotide. The degree of primer homology is a key parameter that indicates the "quality" of a set of primers. Results and discussion. It is established that a significant disadvantage of the vast majority diagnosed using PCR test systems is the lack of internal control of amplification. The negative result of PCR analysis may be due to the absence in the clinical material of a fragment of the Listeria genome, and the fact that the PCR product was not synthesized for other reasons. They may be as the following ones: operator errors, erroneously determined reaction mixture concentrations and PCR temperature parameters. False-negative results can also be caused by factors that inhibit thermostable DNA polymerase. In its turn, such inhibition of the enzyme responsible for amplification is caused by a very large amount of DNA - template, pre-treatment of clinical samples. It has been shown that 80% of clinical specimens contain a substance that inhibits DNA polymerase. Therefore, it is necessary to use internal control, the positive result of the reaction of which indicates the successful amplification, that is the absence of false positive results. Conclusion. There are several reasons why the accuracy of PCR analysis does not reach 100%. Accuracy depends on the technology (variety) of PCR - the method used (ordinary or fluorescent), detection of amplicons, PCR homogeneous or nested, nested in one test tube or in two test tubes, as well as the level of quality of the survey (primarily on the technical parameters of the amplifier). The test systems used can be used for PCR detection and are recommended as standard primer sets for the detection and cross-species testing of listeria, which is important for the timely implementation of appropriate anti-epidemic measures in listeriosis

scholarly journals Can rapid integrated polymerase chain reaction-based diagnostics for gastrointestinal pathogens improve routine hospital infection control practice? A diagnostic study

2014 ◽  
Vol 18 (53) ◽  
pp. 1-167 ◽  
Author(s):  
Louise Pankhurst ◽  
Louissa Macfarlane-Smith ◽  
James Buchanan ◽  
Luke Anson ◽  
Kerrie Davies ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Infection Control ◽  
Sensitivity And Specificity ◽  
Chain Reaction ◽  
Luminex Assay ◽  
Multiplex Assays ◽  
Infectious Diarrhoea ◽  
Gastrointestinal Pathogens ◽  
Microbiological Testing ◽  
Polymerase Chain

BackgroundEvery year approximately 5000–9000 patients are admitted to a hospital with diarrhoea, which in up to 90% of cases has a non-infectious cause. As a result, single rooms are ‘blocked’ by patients with non-infectious diarrhoea, while patients with infectious diarrhoea are still in open bays because of a lack of free side rooms. A rapid test for differentiating infectious from non-infectious diarrhoea could be very beneficial for patients.ObjectiveTo evaluate MassCode multiplex polymerase chain reaction (PCR) for the simultaneous diagnosis of multiple enteropathogens directly from stool, in terms of sensitivity/specificity to detect four common important enteropathogens:Clostridium difficile,Campylobacterspp.,Salmonellaspp. and norovirus.DesignA retrospective study of fixed numbers of samples positive forC. difficile(n = 200),Campylobacterspp. (n = 200),Salmonellaspp. (n = 100) and norovirus (n = 200) plus samples negative for all these pathogens (n = 300). Samples were sourced from NHS microbiology laboratories in Oxford and Leeds where initial diagnostic testing was performed according to Public Health England methodology. Researchers carrying out MassCode assays were blind to this information. A questionnaire survey, examining current practice for infection control teams and microbiology laboratories managing infectious diarrhoea, was also carried out.SettingMassCode assays were carried out at Oxford University Hospitals NHS Trust. Further multiplex assays, carried out using Luminex, were run on the same set of samples at Leeds Teaching Hospitals NHS Trust. The questionnaire was completed by various NHS trusts.Main outcome measuresSensitivity and specificity to detectC. difficile,Campylobacterspp.,Salmonellaspp., and norovirus.ResultsNucleic acids were extracted from 948 clinical samples using an optimised protocol (200Campylobacterspp., 199C. difficile, 60S. enterica, 199 norovirus and 295 negative samples; some samples contained more than one pathogen). Using the MassCode assay, sensitivities for each organism compared with standard microbiological testing ranged from 43% to 94% and specificities from 95% to 98%, with particularly poor performance forS. enterica. Relatively large numbers of unexpected positives not confirmed with quantitative PCR were also observed, particularly forS. enterica,Giardia lambliaandCryptosporidiumspp. As the results indicated thatS. entericadetection might provide generic challenges to other multiplex assays for gastrointestinal pathogens, the Luminex xTag®gastrointestinal assay was also run blinded on the same extracts (937/948 remaining) and on re-extracted samples (839/948 with sufficient material). ForCampylobacterspp.,C. difficileand norovirus, high sensitivities (> 92%) and specificities (> 96%) were observed. ForS. enterica, on the original MassCode/Oxford extracts, Luminex sensitivity compared with standard microbiological testing was 84% [95% confidence interval (CI) 73% to 93%], but this dropped to 46% on a fresh extract, very similar to MassCode, with a corresponding increase in specificity from 92% to 99%. Overall agreement on the per-sample diagnosis compared with combined microbiology plus PCR for the main four/all pathogens was 85.6%/64.7%, 87.0%/82.9% and 89.8%/86.8% for the MassCode assay, Luminex assay/MassCode extract and Luminex assay/fresh extract, respectively. Luminex assay results from fresh extracts implied that 5% of samples did not represent infectious diarrhoea, even though enteropathogens were genuinely present. Managing infectious diarrhoea was a significant burden for infection control teams (taking 21% of their time) and better diagnostics were identified as having major potential benefits for patients.ConclusionsOverall, the Luminex xTag gastrointestinal panel showed similar or superior sensitivity and specificity to the MassCode assay. However, on fresh extracts, this test had low sensitivity to detect a key enteric pathogen,S. enterica; making it an unrealistic option for most microbiology laboratories. Extraction efficiency appears to be a major obstacle for nucleic acid-based tests for this organism, and possibly the whole Enterobacteriaceae family. To improve workflows in service microbiology laboratories, to reduce workload for infection control practitioners, and to improve outcomes for NHS patients, further research on deoxyribonucleic acid-based multiplex gastrointestinal diagnostics is urgently needed.FundingThe Health Technology Assessment programme of the National Institute for Health Research.

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