scholarly journals Development of a Novel Cytochrome b Real-Time PCR Assay for Identification of Plasmodium malariae

2020 ◽  
Vol 36 (3) ◽  
Author(s):  
Dinh Thi Thu Hang ◽  
Vu Thi Nga ◽  
Hoang Van Tong ◽  
Hoang Xuan Su ◽  
Le Quoc Tuan ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Plasmodium Falciparum ◽  
Real Time ◽  
Cytochrome B ◽  
Real Time Pcr ◽  
Clinical Microbiology ◽  
Plasmodium Malariae ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Polymerase Chain

This article aims to establish a novel cytochrome b real-time PCR assay using Taqman probe for identification of P. malariae and its discrimination from other Plasmodium human infecting species. The optimization of real-time PCR assay with 1X QuantiTect Probe PCR Master Mix, primers and probe used at concentrations of 0.4 μM and 0.1 μM, respectively; and 2.5 mM MgCl2, 5 μl DNA template and deionized H2O of 20 μl, was performed using a real-time PCR instrument. The developed real-time PCR assay was evaluated for the limit of detection, stability on standard panels (109-100 copies/ µl), as well as the sensitivity, specificity on control groups. The probit analysis demonstrates that the 95% detection limit was <0.5 parasite/μl, both the sensitivity and specificity of the assay were 100% when evaluated on the control groups.  Additionally, the assay initially evaluated on 41 clinical samples including 21 malaria samples and 20 samples of volunteer blood donors, identified 1 positive sample with P. malariae from the disease group, which shows a concordant result with nested PCR. This novel Cyt b real-time PCR assay for identifying P. malariae may also facilitate earlier discrimination of P. malariae from other Plasmodium parasites with high sensitivity. Keywords Cytochrome b, malaria parasite, plasmodium malariae, mitochondria, real-time PCR. References [1] B. Singh, C. Daneshvar, Human infections and detection of Plasmodium knowlesi, Clinical microbiology reviews 26(2) (2013) 165-84. https://doi.org/10.1128/cmr.00079-12.[2] World Health Organization, Regional and global trends in burden of malaria cases and deaths, World malaria report 2019, Geneva, pp. 4-12.[3] World Health Organization, Progress towards elimination during the RBM decade 2000-2010, Eliminating malaria: learning from the past, looking ahead, Geneva (2011), pp. 39-70.[4] J.M. Vinetz, J. Li, T.F. McCutchan, et al., Plasmodium malariae infection in an asymptomatic 74-year-old Greek woman with splenomegaly, N Engl J Med 338(6) (1998) 367-71. https://doi.org/10.1056/NEJM199802053380605.[5] E. Lo, K. Nguyen, J. Nguyen, et al., Plasmodium malariae Prevalence and csp Gene Diversity, Kenya, 2014 and 2015, Emerg Infect Dis 23(4) (2017) 601-610. https://doi.org/10.3201/eid2304.161245.[6] W.E. Collins, G.M. Jeffery, Plasmodium malariae: parasite and disease, Clinical microbiology reviews 20(4) (2007) 579-92. https://doi.org/10.1128/CMR.00027-07.[7] M. Adams, S.N. Joshi, G. Mbambo, et al., An ultrasensitive reverse transcription polymerase chain reaction assay to detect asymptomatic low-density Plasmodium falciparum and Plasmodium vivax infections in small volume blood samples, Malar J 14 (2015) 520. https://doi.org/10.1186/s12936-015-1038-z.[8] W. Xu, U. Morris, B. Aydin-Schmidt, et al., SYBR Green real-time PCR-RFLP assay targeting the plasmodium cytochrome B gene-a highly sensitive molecular tool for malaria parasite detection and species determination, PloS one 10(3) (2015) e0120210. https://doi.org/10.1371/journal.pone.0120210.[9] E.M. Burd, Validation of laboratory-developed molecular assays for infectious diseases, Clinical microbiology reviews 23(3) (2010) 550-76. https://doi.org/10.1128/CMR.00074-09.[10] G. Snounou, S. Viriyakosol, X.P. Zhu, et al., High sensitivity of detection of human malaria parasites by the use of nested polymerase chain reaction, Molecular and biochemical parasitology 61(2) (1993) 315-20. https://doi.org/10.1016/0166-6851(93)90077-b.[11] C.G. Haanshuus, K. Morch, B. Blomberg, et al., Assessment of malaria real-time PCR methods and application with focus on low-level parasitaemia, PloS one 14(7) (2019) e0218982. https://doi.org/10.1371/journal.pone.0218982.[12] F. Perandin, N. Manca, A. Calderaro, et al., Development of a real-time PCR assay for detection of Plasmodium falciparum, Plasmodium vivax, and Plasmodium ovale for routine clinical diagnosis, Journal of clinical microbiology 42(3) (2004) 1214-9. https://doi.org/10.1128/jcm.42.3.1214-1219.2004.[13] C.E. Oriero, J.P. van Geertruyden, J. Jacobs, et al., Validation of an apicoplast genome target for the detection of Plasmodium species using polymerase chain reaction and loop mediated isothermal amplification, Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases 21(7) (2015) 686 e1-7. https://doi.org/10.1016/j.cmi.2015.02.025.[14] D.F. Echeverry, N.A. Deason, J. Davidson, et al., Human malaria diagnosis using a single-step direct-PCR based on the Plasmodium cytochrome oxidase III gene, Malaria journal 15 (2016) 128. https://doi.org/10.1186/s12936-016-1185-x.[15] P. Li, Z. Zhao, H. Xing, et al., Plasmodium malariae and Plasmodium ovale infections in the China-Myanmar border area, Malaria journal 15(1) (2016) 557. https://doi.org/10.1186/s12936-016-1605-y.[16] E.T.J. Chong, J.W.F. Neoh, T.Y. Lau, et al., Genetic and haplotype analyses targeting cytochrome b gene of Plasmodium knowlesi isolates of Malaysian Borneo and Peninsular Malaysia, Acta tropica 181 (2018) 35-39. https://doi.org/10.1016/j.actatropica.2018.01.018.[17] C. Farrugia, O. Cabaret, F. Botterel, et al., Cytochrome b gene quantitative PCR for diagnosing Plasmodium falciparum infection in travelers, Journal of clinical microbiology 49(6) (2011) 2191-5. https://doi.org/10.1128/JCM.02156-10.[18] C. Wongsrichanalai, M.J. Barcus, S. Muth, et al., A review of malaria diagnostic tools: microscopy and rapid diagnostic test (RDT), The American journal of tropical medicine and hygiene 77(6 Suppl) (2007) 119-27.[19] H.V. Nguyen, P.V.D. Eede, C. van Overmeir, et al., Marked age-dependent prevalence of symptomatic and patent infections and complexity of distribution of human Plasmodium species in central Vietnam, The American journal of tropical medicine and hygiene 87(6) (2012) 989-995. https://doi.org/10.4269/ajtmh.2012.12-0047.  

scholarly journals Deteksi Plasmodium falciparum dengan menggunakan metode real-time polymerase chain reaction di daerah Likupang dan Bitung

2016 ◽  
Vol 4 (1) ◽  
Author(s):  
David C. Tooy ◽  
Janno B. Bernadus ◽  
Angle Sorisi
Keyword(s):  
Polymerase Chain Reaction ◽  
Plasmodium Falciparum ◽  
Real Time ◽  
Real Time Pcr ◽  
18S Rrna ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Negative Results ◽  
Pcr Method ◽  
Polymerase Chain

Abstract: Malaria is one of the most important parasitic disease which is caused by Plasmodium spp. There are approximately 1,2 billion people in the world with high risk of getting malaria. Plasmodium falciparum (P. falciparum) is the cause of tropical malaria or falciparum malaria, and is responsible for most of the mortality rate. Currently, real-time polymerase chain reaction (RT-PCR) is being studied as an alterative of conventional malarian examination. Mangold et al reported that RT-PCR have 94.1% sensitivity and 100% specificity compared to microscopic examination in detecting P. falciparum. The aim of this research is to detect the presence of P. falciparum using RT-PCR in Likupang and Bitung region. This research were using descriptive design to find out the capability of real-time PCR method to detect P. falciparum in Likupang dan Bitung region. The researcher have examined 71 samples which are fulfill the research sample’s criteria. Postive results of P. falciparum found in 18 samples (25,3%) and negative results in 53 samples (74,6%) of total 71 samples with using RT-PCR. No positive results were found in samples from Likupang. There are positive result of P. falciparum in samples from Bitung. It is concluded that RT-PCR method can detect the presence of P. falciparum from the samples obtained from Likupang and Bitung based on the presence of its DNA. This detection efford is done by using 18S rRNA as target gene and ajust specific temperature on the RT-PCR instrument.Keywords: Plasmodium falciparum, Real-time Polymerase Chain Reaction (PCR), DetectionAbstrak: Malaria merupakan salah satu penyakit penting yang disebabkan oleh parasit Plasmodium spp. Kira-kira 1,2 miliar penduduk dunia memiliki risiko tinggi untuk mendapat malaria. Di Indonesia sendiri, terdapat 343.527 kasus terkonfirmasi dan 45 kematian karena malaria. Plasmodium falciparum (P. Falciparum) merupakan penyebab dari malaria tropika atau malaria falsiparum, dan bertanggung jawab atas sebagian besar angka mortalitas. Saat ini Real-Time Polymerase Chain Reaction (RT-PCR) telah banyak diteliti sebagai alternatif dari pemeriksaan malaria. Mangold dkk melaporkan bahwa real-time PCR memiliki nilai sensitivitas 94,1% dan nilai spesifisitas 100% terhadap pemeriksaan mikroskopis dalam mendeteksi P. falciparum. Penelitian bertujuan untuk mendeteksi P. falciparum dengan menggunakan RT-PCR di daerah Likupang dan Bitung. Penelitian ini menggunakan rancangan penelitian deskriptif untuk mengetahui kemampuan metode real-time PCR dalam mendeteksi P. falciparum di daerah Likupang dan Bitung. Tujuan penelitian ini ialah untuk mendeteksi keberadaan P. falciparum dengan menggunakan metode real-time PCR di daerah Likupang dan Bitung. Peneliti memeriksa 71 sampel darah yang memenuhi kriteria sampel penelitian. Hasil positif P. falciparum ditemukan pada 18 sampel (25,3 %) dan hasil negatif pada 53 sampel (74,6 %) dari total 71 sampel dengan menggunakan RT-PCR. Tidak ditemukannya hasil positif P. falciparum pada sampel dari Likupang. Ditemukan hasil positif P. falciparum pada sampel dari Bitung. Simpulan: Metode RT-PCR dapat mendeteksi P. falciparum berdasarkan keberadaan DNA-nya pada sampel yang diperoleh dari daerah Likupang dan Bitung. Deteksi ini berhasil dilakukan dengan menggunakan 18S rRNA sebagai gen target dan pengaturan suhu tertentu pada instrument RT-PCR.Kata kunci: P. falciparum, Real-time Polymerase Chain Reaction (PCR), Detection

scholarly journals Simple and rapid detection of common fetal aneuploidies using peptide nucleic acid probe-based real-time polymerase chain reaction

2022 ◽  
Vol 12 (1) ◽  
Author(s):  
Subeen Hong ◽  
Seung Mi Lee ◽  
Sohee Oh ◽  
So Yeon Kim ◽  
Young Mi Jung ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Peptide Nucleic Acid ◽  
Melting Curve ◽  
Peak Height ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Polymerase Chain

AbstractTo examine the detection performance of a peptide nucleic acid (PNA) probe-based real-time time polymerase chain reaction (PCR) assay to detect common aneuploidies. Using amniotic fluid samples, PNA probe based real-time PCR (Patio DEP Detection Kit; SeaSun Biomaterials, Korea) assay was performed. PNA probe was designed to hybridize to similar sequences located on different segments of target chromosomes (21, 18, and 13) and a reference chromosome. Amplification of target sequences and melting curve analysis was performed. When analyzing the melting curve, the ratio of the peak height of the target and reference chromosome was calculated and determined as aneuploidy if the ratio of peak height was abnormal. All the results from the PNA probe-based real-time PCR and melting curve analyses were compared to those from conventional karyotyping. Forty-two cases with common aneuploidies (24 of trisomy 21, 12 of trisomy 18, and 6 of trisomy 13) and 131 cases with normal karyotype were analyzed. When comparing the karyotyping results, the sensitivity and specificity of the PNA probe-based real-time PCR assay were both 100%. The level of agreement was almost perfect (k = 1.00). PNA real-time PCR assay is a rapid and easy method for detecting common aneuploidies.

Early and simultaneous detection ofNosema bombycis(Microsporidia: Nosematidae), nucleopolyhedrovirus (Baculoviridae), and densovirus (Parvoviridae) by multiplex real-time polymerase chain reaction inBombyx mori(Lepidoptera: Bombycidae)

2017 ◽  
Vol 149 (2) ◽  
pp. 265-275
Author(s):  
Shan Wu ◽  
Yong-Qiang He ◽  
Xing-Meng Lu ◽  
Xiao-Feng Zhang ◽  
Jiang-Bing Shuai ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Early Detection ◽  
Bombyx Mori ◽  
Real Time ◽  
Real Time Pcr ◽  
Simultaneous Detection ◽  
Post Inoculation ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Polymerase Chain

AbstractAn effective multiplex real-time polymerase chain reaction (PCR) assay for the simultaneous detection of three major pathogens,Nosema bombycisNägeli (Microsporidia: Nosematidae),Bombyx morinucleopolyhedrovirus (Baculoviridae: genusAlphabaculovirus) (NPV), andBombyx moridensovirus (Parvoviridae: genusIteravirus) (DNV), in silkworms (Bombyx mori(Linnaeus); Lepidoptera: Bombycidae) was developed in this study. Polymerase chain reaction and real-time PCR tests and basic local alignment search tool searches revealed that the primers and probes used in this study had high specificities for their target species. The ability of each primer/probe set to detect pure pathogen DNA was determined using a plasmid dilution panel, in which under optimal conditions the multiplex real-time PCR assay showed high efficiency in the detection of three mixed target plasmids with a detection limit of 8.5×103copies forN. bombycisandBombyx moriNPV (BmNPV) and 8.5×104copies forBombyx moriDNV (BmDNV). When the ability to detect these three pathogens was examined in artificially inoculated silkworms, our method presented a number of advantages over traditional microscopy, including specificity, sensitivity, and high-throughput capabilities. Under the optimal volume ratio for the three primer/probe sets (3:2:2=N. bombycis:BmNPV:BmDNV), the multiplex real-time PCR assay showed early detection of BmNPV and BmDNV by day 1 post inoculation using DNA templates of the three pathogens in various combinations from individually infected silkworms; the early detection ofN. bombyciswas possible by day 3 post inoculation using the DNA isolated from the midgut ofN. bombycis-infected silkworms.

Optimization of 6-carboxy-X-rhodamine concentration for real-time polymerase chain reaction using molecular beacon chemistry

2007 ◽  
Vol 53 (3) ◽  
pp. 391-397 ◽  
Author(s):  
Gehua Wang ◽  
Erin Becker ◽  
Christine Mesa
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Molecular Beacon ◽  
Quantitative Polymerase Chain Reaction ◽  
Luxs Gene ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Shiga Toxin 2 ◽  
Polymerase Chain

The optimal 6-carboxy-X-rhodamine (ROX) concentration, which is used as a passive reference dye for real-time quantitative polymerase chain reaction (PCR) with molecular beacon chemistry, was determined with the Mx4000™ Multiplex Quantitative PCR System. Additionally, the effects of changing ROX concentrations on PCR reproducibility, Ct values, and efficiency were investigated with this system by using the PCR data obtained from amplification of the Escherichia coli shiga toxin 2 (stx2) gene and the Campylobacter jejuni luxS gene. This study indicated that different ROX concentrations influence many aspects of the real-time PCR reaction. ROX concentration variation could have consequences in the analysis of quantitative data and may lead to erroneous results. This study further indicated that the optimal ROX concentration is 60 nmol/L for real-time PCR, using molecular beacon chemistry for PCR assay of luxS and stx2 genes.

scholarly journals Early Detection of Leifsonia xyli subsp. xyli in Sugarcane Leaves by Real-Time Polymerase Chain Reaction

Plant Disease ◽  
2007 ◽  
Vol 91 (4) ◽  
pp. 430-434 ◽  
Author(s):  
M. P. Grisham ◽  
Y.-B. Pan ◽  
E. P. Richard
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Close Agreement ◽  
Leaf Tissue ◽  
Conventional Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Polymerase Chain ◽  
Pcr Assays

A real-time, polymerase chain reaction (PCR) assay was developed for detecting Leifsonia xyli subsp. xyli in sugarcane leaf tissue. Real-time PCR assays were conducted on the youngest, fully expanded leaf of three cultivars collected bi-weekly from field nurseries between 11 April and 19 July 2005. L. xyli subsp. xyli infection was detected in leaves collected at all sampling dates, including those from 1-month-old plants on 11 April. Assays conducted on older, more rapidly growing plants (28 July and 21 October 2005) indicated that leaf position affects assay efficiency. Conventional PCR was less efficient than real-time PCR for detecting L. xyli subsp. xyli in leaf tissue. Real-time PCR was used to rank cultivars for susceptibility to L. xyli subsp. xyli infection based on the relative titer of L. xyli subsp. xyli in leaves of inoculated, 3- and 4-month-old greenhouse-grown plants. The ranking of cultivars by real-time PCR was in close agreement with the ranking determined by tissue-blot enzyme immunoassay performed on tissue from 7- to 9-month-old stalks.

scholarly journals Perbandingan deteksi Plasmodium falciparum dengan metode pemeriksaan mikroskopik dan teknik real-time polymerase chain reaction

2016 ◽  
Vol 4 (1) ◽  
Author(s):  
Elril T. Langi ◽  
Janno B. B. Bernadus ◽  
Greta J. P. Wahongan
Keyword(s):  
Polymerase Chain Reaction ◽  
Plasmodium Falciparum ◽  
Real Time ◽  
Real Time Pcr ◽  
Gold Standard ◽  
Dna Amplification ◽  
World Health ◽  
Chain Reaction ◽  
Blood Samples ◽  
Polymerase Chain

Abstract: Plasmodium falciparum is one of the species of parasites causing tropical malaria disease. Plasmodium falciparum was reported as often being the major source of pain and even death in most cases. The data released by WHO shows that, globally, 198 millions of malaria cases occurred in 2013 with 548 thousands as cause of death. Microscopic examination is a gold standard for detecting Plasmodium falciparum. Although this method has certain limitations in diagnosing complication infection, phases of parasitemia, and also the capability of laboratory's medical staff factor. Nowadays, there has been innovation in biomolecular department, that is examination using PCR which can accurately detect the plasmodium, due to the DNA amplification. This method however, has not often used by doctors in diagnose malaria disease. The aim of this research is to determine the comparison of malaria detection using microscopic verification of plasmodium falciparum with real-time PCR verification. The method used in this research is diagnostic with 35 blood samples of patient suffering malaria disease. The blood samples from patient's vena were then divided into thick and thin microscopic sample, and some were putted into EDTA tube for DNA extraction in the laboratory using real-time PCR verification. The result of this research shown that sensitivity and specificity rate of PCR is 100% accurate. Conclusion: detection result of plasmodium falciparum using real-time PCR verification produced equal result as microscopic verification.Keywords: Plasmodium falciparum, Microscopic method, Real-time Polymerase Chain Reaction (PCR)Abstrak: Plasmodium falciparum adalah salah satu spesies parasit penyebab penyakit malaria, yaitu malaria tropika. Plasmodium falciparum dilaporkan sebagai spesies yang paling banyak menyebabkan angka kesakitan dan kematian pada manusia akibat penyakit malaria. World Health Organization (WHO) melaporkan secara global, diperkirakan 198 juta kasus malaria terjadi secara keseluruhan pada tahun 2013 dan menyebabkan 584 ribu kematian. Pemeriksaan mikroskopik adalah pemeriksaan gold standard untuk mendeteksi Plasmodium falciparum. Namun pemeriksaan ini memiliki keterbatasan dalam hal mendiagnosis infeksi campuran, infeksi dengan keadaan parasitemia, dan tidak terlatihnya tenaga kesehatan laboratorium. Saat ini dalam bidang biomolekuler telah dikembangkan pemeriksaan real-time polymerase chain reaction (PCR) yang akurat untuk mendeteksi plasmodium, karena didasarkan pada amplifikasi DNA plasmodium, namun pemeriksaan ini belum rutin digunakan untuk mendiagnosis malaria. Penelitian ini bertujuan untuk mengetahui perbandingan deteksi Plasmodium falciparum dengan pemeriksaan mikroskopik dan pemeriksaan real-time PCR. Metode penelitian ini ialah uji diagnostik. Sampel pada penelitian ini yaitu 35 sampel darah pasien suspek malaria. Sampel darah vena yang diambil langsung dibuat sedian darah tipis dan sediaan darah tebal untuk diperiksa di mikroskop, sedangkan darah yang tersisa dimasukkan dalam tabung EDTA, dan dibawa ke Laboratorium untuk dibuat ekstraksi DNA dan dilanjutkan dengan pemeriksaan real-time PCR. Hasil penelitian menunjukkan tingkatsensitivitas dan spesifisitas real-time PCR sebesar 100%. Simpulan: Hasil deteksi Plasmodium falciparum dengan pemeriksaan real-time PCR memiliki efektivitas yang setara dengan metode pemeriksaan mikroskopik sebagai gold standart.Kata kunci: Plasmodium falciparum, Pemeriksaan Mikroskopik, Real-time Polymerase Chain Reaction (PCR)

scholarly journals Validation of a Real-Time Polymerase Chain Reaction Assay for the Identification of Meloidogyne arenaria

Plant Disease ◽  
2011 ◽  
Vol 95 (7) ◽  
pp. 835-838 ◽  
Author(s):  
Paula Agudelo ◽  
Stephen A. Lewis ◽  
Bruce A. Fortnum
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Soil Samples ◽  
Meloidogyne Arenaria ◽  
Chain Reaction ◽  
Pcr Assay ◽  
The Real ◽  
Polymerase Chain ◽  
Species Specific

Meloidogyne arenaria is an economically important parasite of many crops worldwide. Identification and detection of this species in soil samples is necessary for the design of crop rotation systems, selection of resistant cultivars, and potential use of biological control options. The objective of this study was to develop and validate a real-time polymerase chain reaction (PCR) assay, using species-specific primers and SYBR Green I Dye, for identification of M. arenaria. The specificity of the assay was confirmed by testing for amplification of DNA from other Meloidogyne spp. and from M. arenaria populations of different geographic origins. Field soil samples containing a mixture of M. arenaria and M. incognita were used to compare identification by the real-time PCR assay with identification by esterase phenotype analysis of mature females and by morphometrics of juveniles. The real-time PCR assay provided an accurate and sensitive means for the identification of single juveniles from soil samples.

scholarly journals Identification and Detection of Phoma tracheiphila, Causal Agent of Citrus Mal Secco Disease, by Real-Time Polymerase Chain Reaction

Plant Disease ◽  
2006 ◽  
Vol 90 (12) ◽  
pp. 1523-1530 ◽  
Author(s):  
G. Licciardello ◽  
F. M. Grasso ◽  
P. Bella ◽  
G. Cirvilleri ◽  
V. Grimaldi ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Causal Agent ◽  
Conventional Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Polymerase Chain ◽  
Phoma Tracheiphila ◽  
Mal Secco

Phoma tracheiphila is the causal agent of a tracheomycotic disease of citrus called mal secco causing the dieback of twigs and branches. This pathogen is of quarantine concern; therefore, fast and reliable protocols are required to detect it promptly. A specific primer pair and a dual-labeled fluorogenic probe were used in a real-time polymerase chain reaction (PCR) with the Cepheid Smart Cycler II System (Transportable Device TD configuration) to detect this fungus in citrus samples. Real-time PCR assay was compared to modified conventional PCR assay. The sensitivity of the former was evaluated by testing P. tracheiphila DNA dilutions, and the minimum amount detectable was about 500 fg, whereas the linear quantification range was within 100 ng to 1 pg. Conventional PCR sensitivity was 10 pg. Conventional and real-time PCR successfully detected the fungus in woody samples of naturally infected lemon and artificially inoculated sour orange seedlings. Nevertheless, real-time PCR was about 10- to 20-fold more sensitive than conventional PCR, and preliminary results indicate that the former technique achieves quantitative monitoring of the fungus in tissues. Simple and rapid procedures to obtain suitable DNA samples from fungal cultures and citrus woody samples for PCR assays enable diagnosis to be completed in a short time.

scholarly journals Sensitive Detection of Fusarium circinatum in Pine Seed by Combining an Enrichment Procedure with a Real-Time Polymerase Chain Reaction Using Dual-Labeled Probe Chemistry

2009 ◽  
Vol 99 (5) ◽  
pp. 582-590 ◽  
Author(s):  
Renaud Ioos ◽  
Céline Fourrier ◽  
Gabriela Iancu ◽  
Thomas R. Gordon
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Fusarium Circinatum ◽  
Conventional Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
The Real ◽  
Polymerase Chain ◽  
Seed Dna

Fusarium circinatum is the causal agent of pitch canker disease on numerous Pinus spp. This aggressive fungus may infect pine seed cryptically and, therefore, can easily be spread long distances by the seed trade. F. circinatum has recently been listed as a quarantine organism in numerous countries throughout the world, which prompted the development of a specific and sensitive tool for the detection of this pathogen in conifer seed. A new detection protocol for F. circinatum based on a biological enrichment step followed by a real-time polymerase chain reaction (PCR) assay was developed. Several enrichment protocols were compared and a 72-h incubation of the seed with potato dextrose broth was the most efficient technique to increase F. circinatum biomass before DNA extraction. The relative accuracy, specificity, and sensitivity of the real-time PCR assay was evaluated in comparison with a previously published conventional PCR test on 420 seed DNA extracts. The real-time PCR described here proved to be highly specific and significantly more sensitive than the conventional PCR, and enabled the detection of F. circinatum in samples artificially contaminated with less than 1/1,000 infected seed, as well as in naturally infected samples. Last, in order to routinely check the quality of the seed DNA extracts, a primer–probe combination that targets a highly conserved region within the 18S ribosomal DNA in plants or fungi was successfully developed. This assay allows for quick and reliable detection of F. circinatum in seed, which can help to prevent long-distance spread of the pathogen via contaminated seed lots.

scholarly journals Development of Real-Time Polymerase Chain Reaction Assay with TaqMan Probe for the Quantitative Detection of Infectious Hypodermal and Hematopoietic Necrosis Virus from Shrimp

2006 ◽  
Vol 89 (1) ◽  
pp. 240-244 ◽  
Author(s):  
Zhi-Qin Yue ◽  
Hong Liu ◽  
Wei-Ji Wang ◽  
Zhi-Wen Lei ◽  
Cheng-Zhu Liang ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Taqman Probe ◽  
Quantitative Detection ◽  
Necrosis Virus ◽  
Chain Reaction ◽  
Pcr Assay ◽  
The Real ◽  
Polymerase Chain

Abstract An assay was developed for the detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) based on real-time quantitative polymerase chain reaction (PCR). A pair of primers and a TaqMan probe were designed that are specific for the recognition of a conservative region in the IHHNV genome. The IHHNV real-time PCR assay had a detection limit of 9 DNA copies,with a dynamic range of detection between 9 106 and 9 DNA copies. The primer pairs and probe were specific to IHHNV and did not cross-reactwith shrimp genomic DNAor other shrimp viruses such as White Spot Syndrome Virus (WSSV), Monodon Baculovirus (MBV), and hepatopancreatic parvovirus (HPV). This assay has a broad application for basic and clinical investigations. For clinical samples, the real-time PCR assay detected all the positive samples screened by conventional PCR, which indicated the sensitivity of the real-time assay. The IHHNV real-time PCR assay with high sensitivity, specificity, wide range of detection ability, and simplicity is particularly useful for screening large numbers of specimens and measuring viral loads to monitor the broodstock.

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