Molecular diagnostics in clinical parasitology and mycology: limits of the current polymerase chain reaction (PCR) assays and interest of the real-time PCR assays

The rickettsia Anaplasma marginale is considered the main agent of bovine anaplasmosis. Due the nonspecific clinical signs of the anaplasmosis, the diagnosis of infection depends of laboratory confirmation. In recent years, molecular diagnostic methods have been used to detect A. marginale in cattle. However, the existence of a large number of assays of different sensitivity and cost makes the choice of an appropriate test difficult. In the present study, a real-time Polymerase Chain Reaction (PCR) based on the msp5 target gene was quantitatively assessed and compared to an end point PCR. Both reactions were subjected to sensitivity and specificity evaluation using plasmid DNA and samples from cattle experimentally infected with A. marginale. A comparative field trial of the tests was carried out using samples of cattle from a stable enzootic area for A. marginale. The real-time PCR showed a higher sensitivity than the end point PCR. This reaction (i.e. real-time PCR) was able to detect one copy of the msp5 gene in 100 ηg of plasmidial DNA, and more than 80% of its results were positive among experimentally infected animals seven days after infection. In addition, based on in silico analysis, the real-time PCR evaluated in the present study appears to be useful for the detection of A. ovis.

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Quantification of Magnaporthe grisea During Infection of Rice Plants Using Real-Time Polymerase Chain Reaction and Northern Blot/Phosphoimaging Analyses

Phytopathology ◽

10.1094/phyto.2002.92.8.870 ◽

2002 ◽

Vol 92 (8) ◽

pp. 870-876 ◽

Cited By ~ 88

Author(s):

Min Qi ◽

Yinong Yang

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Northern Blot ◽

Host Resistance ◽

Real Time Pcr ◽

Magnaporthe Grisea ◽

Chain Reaction ◽

Fungal Pathogenicity ◽

The Real ◽

Polymerase Chain

Rice blast, caused by Magnaporthe grisea, is a serious fungal disease of rice worldwide. Currently, evaluation of the fungal pathogenicity and host resistance is mainly based on a disease rating or measurement of blast lesion number and size. However, these methods only provide visual estimation rather than accurate measurement of fungal growth in rice plants. In this study, DNA-based real-time polymerase chain reaction (PCR) and RNA-based northern blot/phosphoimaging analyses were evaluated to quantify M. grisea. Both methods were sensitive, specific, and reproducible and could accurately measure the relative growth and absolute biomass of M. grisea. The real-time PCR analysis showed that the growth of M. grisea in seedling leaves of susceptible cultivars (M201 and Wells) was ≈46 to 80 times higher than that of a resistant cultivar (Drew) at 4 and 6 days after inoculation. The data obtained from the real-time PCR assays also were consistent with that from northern blot/ phosphoimaging analysis. However, the real-time PCR approach was much faster and more convenient in most cases. Therefore, it is an excellent tool for in planta quantification of M. grisea and can be used for reliable assessment of fungal pathogenicity and host resistance

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SLCO1B1 c.521T>C Genotyping in the Austrian Population Using 2 Commercial Real-Time Polymerase Chain Reaction Assays: An Implementation Study

Pharmacology ◽

10.1159/000490619 ◽

2018 ◽

Vol 102 (1-2) ◽

pp. 88-90

Author(s):

Dietmar Enko ◽

Sophia Harringer ◽

Christian Oberkanins ◽

Helene Pühringer ◽

Gabriele Halwachs-Baumann ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Real Time Pcr ◽

Clinical Performance ◽

Chain Reaction ◽

Increased Risk ◽

Polymerase Chain ◽

Pcr Assays ◽

Austrian Population ◽

Genotype And Allele Frequencies

Statin-induced myopathy is reported to be significantly associated with the SCLO1B1 c.521T>C polymorphism. To date, SLCO1B1 c.521T>C epidemiologic data for the Austrian population is still lacking. Therefore, this study aimed at assessing the genotype and allele frequencies of the SLCO1B1 c.521T>C variant in Austria and evaluating the clinical performance of 2 commercial real-time polymerase chain reaction (PCR) assays. Genomic DNA isolated from 181 healthy individuals was analyzed for the SLCO1B1 c.521T>C polymorphism in a comparative manner using the SLCO1B1 c.521T>C RealFastTM Assay and the BioPro SLCO1B1 Genotyping real-time PCR Kit. A total of 10 (5.5%) and 44 (24.3%) out of 181 individuals were SLCO1B1 c.521T>C C/C-homo- and C/T-heterozygotes, the genotypes indicative of high and increased risk of statin-induced myopathy, respectively. The SLCO1B1 c.521C allele frequency rate was 17.7%. In conclusion, the genetic predisposition of elevated statin-induced myopathy risk in the Austrian population is frequent. Both real-time PCR assays under investigation here are reliable and robust SLCO1B1 c.521T>C genotyping tools in clinical routine.

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

Plant Disease ◽

10.1094/pdis-91-4-0430 ◽

2007 ◽

Vol 91 (4) ◽

pp. 430-434 ◽

Cited By ~ 27

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.

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

Plant Disease ◽

10.1094/pdis-09-10-0668 ◽

2011 ◽

Vol 95 (7) ◽

pp. 835-838 ◽

Cited By ~ 6

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.

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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

Phytopathology ◽

10.1094/phyto-99-5-0582 ◽

2009 ◽

Vol 99 (5) ◽

pp. 582-590 ◽

Cited By ~ 60

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.

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Development and evaluation of a real-time polymerase chain reaction for fast diagnosis of sporotrichosis caused by Sporothrix globosa

Medical Mycology ◽

10.1093/mmy/myz029 ◽

2019 ◽

Vol 58 (1) ◽

pp. 61-65 ◽

Cited By ~ 1

Author(s):

Mingrui Zhang ◽

Fuqiu Li ◽

Jie Gong ◽

Xin Yang ◽

Jianzhong Zhang ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Real Time Pcr ◽

Specific Method ◽

Chain Reaction ◽

Clinical Specimens ◽

Sporothrix Globosa ◽

The Real ◽

Pcr Method ◽

Polymerase Chain

Abstract Sporothrix globosa is an important clinical pathogen in the Sporothrix complex, which is causing sporotrichosis. S. globosa is distributed worldwide, especially in Asia. The transmission medium of S. globosa is mainly contaminated soil or decaying vegetation, and the infection usually caused by transcutaneous trauma, through which the fungal conidia or yeast cells enter the host. Although the clinical manifestations of sporotrichosis caused by S. globosa is always benign, there have been several outbreaks worldwide. In this study, we established a novel real-time polymerase chain reaction (PCR) method based on the internal transcribed spacer (ITS) sequence for the identification of S. globosa. The assay was further evaluated by clinical specimens obtained from patients of sporotrichosis. The sensitivity and specificity of the real-time PCR method was both 100%. The detection limit was 10 fg. The positive detection rate for 30 clinical specimens, which were confirmed infected by S. globosa, was 100%. The real-time PCR method established in this paper is a rapid, sensitive and specific method for the identification of S. globosa. It can detect S. globosa in clinical specimen from patients with sporotrichosis, which is helpful for fast clinical diagnosis.

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

Journal of AOAC International ◽

10.1093/jaoac/89.1.240 ◽

2006 ◽

Vol 89 (1) ◽

pp. 240-244 ◽

Cited By ~ 5

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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Genotyping Common Bean for the Potyvirus Resistance Alleles I and bc-12 with a Multiplex Real-Time Polymerase Chain Reaction Assay

Phytopathology ◽

10.1094/phyto-95-0499 ◽

2005 ◽

Vol 95 (5) ◽

pp. 499-505 ◽

Cited By ~ 12

Author(s):

George J. Vandemark ◽

Phillip N. Miklas

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Real Time Pcr ◽

Probability Distributions ◽

Reference Sample ◽

Bean Common Mosaic Virus ◽

Progeny Testing ◽

Chain Reaction ◽

Polymerase Chain ◽

Pcr Assays

A multiplex real-time polymerase chain reaction (PCR) assay was developed to simultaneously genotype plants for the I and bc-12 alleles, which condition resistance in beans to Bean common mosaic virus and Bean common mosaic necrosis virus. A segregating F2 population was derived from the cross between pinto bean breeding line P94207-189A (bc-1 bc-1 I I) and Olathe (bc-12 bc-12 i i). Real-time PCR assays were developed that were specific for each allele, and a multiplex PCR reaction could unambiguously assign F2 plants to one of nine genotypes. Remnant F1 plants were used as a comparative reference sample. PCR results among this sample fit a normal distribution for both real-time PCR assays, and 99% probability distributions were determined for heterozygotes. F2 plants were genotyped based on results relative to the probability distributions for heterozygotes. F2 plants also were genotyped for the I and bc-12 alleles by performing F3 family progeny tests for virus resistance. Agreement between the two methods was 100% (198/198) for the bc-12 allele, and 92.4% (183/198) for the I allele. Erroneous genotyping was due to recombination between the amplicon and the I allele. Realtime PCR assays provide a robust method for genotyping seedlings and, in some cases, may eliminate the need for progeny testing.

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