3′ RACE LaNe: a simple and rapid fully nested PCR method to determine 3′-terminal cDNA sequence

AbstractAcanthamoeba spp. are opportunistic human pathogens that cause granulomatous amoebic encephalitis and keratitis, and their accurate detection and enumeration in environmental samples is a challenge. In addition, information regarding the genotyping of Acanthamoeba spp. using various PCR methods is equally critical. Therefore, considering the diverse niches of habitats, it is necessary to develop an even more efficient genotyping method for Acanthamoeba spp. detection. This study improved the sensitivity of detection to avoid underestimation of Acanthamoeba spp. occurrence in aquatic environmental samples, and to accurately define the pathogenic risk by developing an efficient PCR method. In this study, a new nested genotyping method was established and compared with various PCR-based methods using in silico, lab, and empirical tests. The in silico test showed that many PCR-based methods could not successfully align specific genotypes of Acanthamoeba, except for the newly designed nested PCR and real-time PCR method. Furthermore, 52 water samples from rivers, reservoirs, and a river basin in Taiwan were analysed by six different PCR methods and compared for genotyping and detection efficiency of Acanthamoeba. The newly developed nested-PCR-based method of genotyping was found to be significantly sensitive as it could effectively detect the occurrence of Acanthamoeba spp., which was underestimated by the JDP-PCR method. Additionally, the present results are consistent with previous studies indicating that the high prevalence of Acanthamoeba in the aquatic environment of Taiwan is attributed to the commonly found T4 genotype. Ultimately, we report the development of a small volume procedure, which is a combination of recent genotyping PCR and conventional real-time PCR for enumeration of aquatic Acanthamoeba and acquirement of biologically meaningful genotyping information. We anticipate that the newly developed detection method will contribute to the precise estimation, evaluation, and reduction of the contamination risk of pathogenic Acanthamoeba spp., which is regularly found in the water resources utilised for domestic purposes.

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Estimating Giardia cyst viability using RT-PCR

Water Science & Technology Water Supply ◽

10.2166/ws.2002.0092 ◽

2002 ◽

Vol 2 (3) ◽

pp. 107-115 ◽

Cited By ~ 1

Author(s):

M. Maux ◽

I. Bertrand ◽

C. Gantzer ◽

J. Schwartzbrod

Keyword(s):

Nested Pcr ◽

Target Genes ◽

Molecular Techniques ◽

Giardia Cyst ◽

Health Issues ◽

Structural Protein ◽

Gene Encoding ◽

Response To Stress ◽

Pcr Method ◽

Giardia Cysts

The aim of this work was to evaluate the use of molecular techniques for the detection of viable Giardia cysts in the environment to assess public health issues. Three target genes were selected: the heat shock protein gene, HSP70, which is expressed in response to stress; the giardin gene, which encodes a structural protein; and, alcohol dehydrogenase E (ADHE), a novel gene encoding an enzyme involved in the metabolism of energy. We tested the efficiency of five protocols for the extraction of either genomic DNA or total RNA from Giardia cysts: two of these protocols were previously cited in the literature and three consisted of commercial DNA extraction kits. The brands of enzyme were determined according to the primers chosen and the amplification conditions were optimised: 2.5 mM MgCl2, 0.5 mM primers and 60°C for annealing temperature. A semi-nested PCR method and an RT semi-nested PCR procedure were developed to detect mRNA from these three genes and to estimate the viability of Giardia cysts.

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Use of a nested PCR method for the detection of astrovirus serotype 1 in human faecal material

Molecular and Cellular Probes ◽

10.1006/mcpr.1994.1069 ◽

1994 ◽

Vol 8 (6) ◽

pp. 481-486 ◽

Cited By ~ 5

Author(s):

M. Shi ◽

S. Sikotra ◽

T. Lee ◽

J.B. Kurtz ◽

B. Getty ◽

...

Keyword(s):

Nested Pcr ◽

Faecal Material ◽

Serotype 1 ◽

Pcr Method

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Development of Ultra-rapid Nested PCR Method for Detection of Specific Gene of Tracheal Mite (Acarapis woodi)

Journal of Apiculture ◽

10.17519/apiculture.2019.04.34.1.15 ◽

2019 ◽

Vol 34 (1) ◽

pp. 15-26

Author(s):

MoonJung Kim ◽

Byoung-Hee Kim ◽

SoMin Kim ◽

Truong A Tai ◽

Jung-Min Kim ◽

...

Keyword(s):

Nested Pcr ◽

Specific Gene ◽

Acarapis Woodi ◽

Tracheal Mite ◽

Pcr Method

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Evaluation of Mycoplasma arginini abundance in slaughtered camels with pneumonia confirm with nested PCR method for the first time in Iran

Clinical Biochemistry ◽

10.1016/j.clinbiochem.2011.08.757 ◽

2011 ◽

Vol 44 (13) ◽

pp. S300

Author(s):

Izadi Maryam ◽

Navid Mehr Jafar ◽

Lal Alizadeh Samane ◽

Bolori Mehrin ◽

Bahmani Somaye

Keyword(s):

Nested Pcr ◽

Pcr Method ◽

First Time

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An evaluation of the usefulness of invasive and non-invasive methods used to diagnose Helicobacter spp. infections in dogs

Polish Journal of Veterinary Sciences ◽

10.1515/pjvs-2017-0059 ◽

2017 ◽

Vol 20 (3) ◽

pp. 491-499 ◽

Cited By ~ 2

Author(s):

M. Jankowski ◽

J. Spużak ◽

K. Kubiak ◽

K. Glińska-Suchocka ◽

M. Biernat

Keyword(s):

Nested Pcr ◽

Gastric Biopsy ◽

Diagnostic Methods ◽

Rapid Urease Test ◽

Pcr Assay ◽

Non Invasive ◽

Biopsy Specimens ◽

Urease Test ◽

Invasive Method ◽

Pcr Method

AbstractThe aim of this study was to assess the suitability of invasive and non-invasive methods used to diagnose Helicobacter spp. in the stomachs of dogs. The study was carried out on 30 dogs of both sexes and different breeds, between one and 15 years old. A histopathologic examination, a microbiological culture, a rapid urease test, a direct bacteriological preparation and a nested PCR assay were carried out. Gastric Helicobacter spp. was identified in gastric biopsy specimens from 16 (53.3%) dogs using direct bacteriological preparation, in four (13.3%) dogs based on a culture, in 23 (76.6%) dogs using the rapid urease test and in 21 (70,0%) dogs based on a histopathological assessment of the biopsy specimens. The nested PCR of the gastric biopsy specimens revealed gastric Helicobacter spp. in all the dogs (100%). A saliva PCR assay revealed gastric Helicobacter spp. in 23 (76.6%) dogs, while stool PCR revealed the bacterium in seven (23.3%) dogs. We found that invasive methods were more accurate than non-invasive methods in detecting a Helicobacter spp. infection in dogs. In addition, the nested PCR method used to evaluate the gastric mucosal biopsy specimens was the most accurate test for detecting Helicobacter spp. It was further found that the PCR-based saliva assay was the best non-invasive method for detecting Helicobacter spp. However, taking into consideration that most of the diagnostic methods used to detect this bacterium have drawbacks, at least two diagnostic methods should be used to detect Helicobacter spp. as is done in human medicine.

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KARAKTERISASI FITOPLASMA PENYEBAB PENYAKIT LAYU KELAPA DI PULAU DERAWAN MENGGUNAKAN RFLP IN SILICO

JURNAL HAMA DAN PENYAKIT TUMBUHAN TROPIKA ◽

10.23960/j.hptt.217105-110 ◽

2018 ◽

Vol 17 (2) ◽

pp. 105

Author(s):

Agus Eko Prasetyo ◽

Kikin Hamzah Mutaqin ◽

Giyanto .

Keyword(s):

Nested Pcr ◽

In Silico ◽

Bacterial Species ◽

Rflp Analysis ◽

Wilt Disease ◽

Rrna Gene ◽

16S Rrna Gene Sequences ◽

Gram Positive Bacteria ◽

Pcr Method

Characterization of phytoplasmas associated with coconut wilt disease in Derawan Island using in silico RFLP. Coconutwilt disease has been reported in Derawan Island that resulted in eradication up to 10% of the total cultivated palms. Theobjective of this study was to detect and characterize phytoplasmas associated with coconut wilt disease in Derawan islandusing nested PCR technique and in silico RFLP based on 16S rRNA gene sequences. Detection of phytoplasmas was performedusing nested PCR method, cloning of nPCR products, sequencing, and analysis of sequencing results using in silico RFLP.The results revealed that phytoplasmas could not be detected by PCR using P1/P7 primer pairs however it could be amplifiedby nested PCR using R16F2n/R16R2 primer pairs resulting amplicon at about 1.25 kb. In silico RFLP analysis indicated thatphytoplasmas associated with coconut wilt disease in Derawan Island belong to 16SrII (witches broom phytoplasma). PCRproduct of the nPCR need to be sequenced because the R16F2n/R16R2 primer will also amplify the other bacterial species, mainly from Gram positive bacteria.

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Frequency of Chlamydia trachomatis Infection in Spontaneous Abortion of Infertile Women During First Pregnancy Referred to Tabriz University of Medical Sciences by Nested PCR Method in 2015

International Journal of Women s Health and Reproduction Sciences ◽

10.15296/ijwhr.2019.87 ◽

2018 ◽

Vol 7 (4) ◽

pp. 526-230

Author(s):

Mehdi Haghdoost ◽

Sanaz Mousavi ◽

Mehdi Khanbabayi Gol ◽

Majid Montazer

Keyword(s):

Spontaneous Abortion ◽

Nested Pcr ◽

Control Method ◽

Transmitted Disease ◽

Infertility Treatment ◽

P Value ◽

Medical Sciences ◽

Infertile Women ◽

Significant Difference ◽

Pcr Method

Objectives:Chlamydia trachomatis, as the main cause of bacterial sexually transmitted disease (STD), can lead to serious complications such as spontaneous abortion. Therefore, the aim of this study was to determine the frequency of C. trachomatis infection in spontaneous abortion of infertile women who referred to Tabriz University of Medical Sciences during the first pregnancy by means of nested polymerase chain reaction (PCR) method in 2015. Materials and Methods: The present descriptive, cross-sectional study was performed in the infertility clinics of Tabriz University of Medical Sciences from March 21, 2015 to March 19, 2016. A total of 120 infertile women were selected by the convenience sampling method. The specimens were prepared by the Dacron swab after four rotations in the endocervix and discharged into the specific transport medium of C. trachomatis. The DNA extraction was then performed by AccuPrep genomic DNA kit and the DNA was extracted until performing the PCR at -20° C. Next, nested PCR was conducted in 2 rounds and the final product of PCR was agar -2% gel electrophoresis. After entering the data in SPSS, the chi-square test was used to examine the role of factors influencing C. trachomatis infection and a P value less than 0.05 was considered significant. Results: The incidence of C. trachomatis infection in women with spontaneous abortion was 16.66%. In addition, there was a significant difference between the infected and non-infected groups regarding employment (P<0.04), birth control method (P<0.03), and the number of sexual intercourses per week (P<0.001). Conclusions: The prevalence of C. trachomatis in women who became pregnant with infertility treatment and spontaneous abortion was high in this study. Thus, nested PCR is considered an appropriate method for the diagnosis of C. trachomatis and it is essential for pregnant women who experience pregnancy with infertility treatment.

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DETECTION AND IDENTIFICATION OF YELLOW MOSAIC STUNT DISEASE ON Petunia sp. USING NESTED PCR METHOD

JURNAL HAMA DAN PENYAKIT TUMBUHAN TROPIKA ◽

10.23960/jhptt.12156-62 ◽

2021 ◽

Vol 21 (1) ◽

pp. 56-62

Author(s):

Suryani Titi Astuti ◽

Sri Sulandari ◽

Sedyo Hartono ◽

Susamto Somowiyarjo

Keyword(s):

Electron Microscope ◽

Nested Pcr ◽

Molecular Detection ◽

Ornamental Plant ◽

Mechanical Transmission ◽

Chenopodium Amaranticolor ◽

Virus Particles ◽

Central Java ◽

Detection And Identification ◽

Pcr Method

Detection and identification of yellow mosaic stunt disease on Petunia sp. using nested PCR method. Yellow mosaic stuntdisease was found at some nurseries of Petunia in Sleman, Yogyakarta, also in Muntilan and Magelang Central Java. Thedisease was very important due to its ability reducing the quality and quantity of Petunia seedlings. The causal agent of thedisease may be carried over to imported seeds and necessary to identify as a basic information for developing controlstrategies. This research was done by mechanical transmission on indicator plants. The observation of the causal agents wasconducted using electron microscope with quick dipping method and the molecular detection was done using nested PCRwith TobRT up1-TobRT do2 as the external primers and TobN up3-TobN do4 as the internal primers. Mechanical inoculationshowed chlorosis symptoms that developed into local spot on Chenopodium amaranticolor as well as mosaic and veinbanding on Nicotiana benthamiana. The observation using electron microscope showed rod-shaped virus particles sizedapproximately 300 nm and by PCR method produced around 568 bp and 400 bp DNA band. Based on the sequence analysis,the disease was caused by Rehmania mosaic virus. This type of Tobamovirus has 96% similarity with ReMV-Japan. ReMV, aplant pathogen which was a member of Tobamovirus that has never been reported in Indonesia. This research was the firstreport of ReMV in Indonesia infecting Petunia as ornamental plant.

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The role of Helicobacter cinaedi in the development of atherosclerosis

Postępy Higieny i Medycyny Doświadczalnej ◽

10.2478/ahem-2021-0003 ◽

2021 ◽

Vol 75 (1) ◽

pp. 529-536

Author(s):

Tevhide Ziver Sarp ◽

Safa Gode ◽

Suat Saribas ◽

Sevgi Ergin ◽

Gökhan Kasnak ◽

...

Keyword(s):

High Density Lipoprotein ◽

Total Cholesterol ◽

Patient Group ◽

Nested Pcr ◽

Density Lipoprotein ◽

High Density ◽

Control Groups ◽

Helicobacter Cinaedi ◽

Pcr Method ◽

Atherosclerosis Development

Abstract Helicobacter cinaedi (H. cinaedi) is a Gram-negative curved motile rod that causes bloodstream or enteric infections. It was suggested that H. cinaedi was involved in the progression of atherosclerosis. We aimed to investigate the presence of H. cinaedi DNA using a nested-polymerase chain reaction (PCR) in atheroma plaques from patients with atherosclerosis-induced vascular diseases. A total of 129 patients diagnosed with valvular heart disease due to atherosclerosis and 146 patients with non-atherosclerotic post-stenotic dilatation were included as the patient and the control groups, respectively. The ATCC BA847 H. cinaedi strain was used as the positive control for the nested-PCR method. We investigated H. cinaedi DNA in our study groups using the nested-PCR method and detected only six H. cinaedi DNA (4.65%) in the 129 atherosclerotic patient group. We detected significant difference between patient and control groups with respect to the presence of H. cinaedi on the basis of Fischer’s exact test (p = 0.010) by univariate analysis. Age (OR: 1.042, p = 0.016), total cholesterol (≥200 mg/dL) (OR: 1.849, p = 0.0001), and high-density lipoprotein (≥50 mg/dL) (OR: 0.745, p = 0.039) levels were detected as independent variables for the risk of atherosclerosis development in the patient group. The presence of H. cinaedi was not detected as an independent variable in a multivariate analysis. Previous studies suggested that H. cinaedi-induced oral infections might translocate to vascular tissue and induce chronic inflammation in the aorta, which subsequently may lead to atherosclerotic plaque formation. In conclusion, we could not suggest that there is a causal relationship between H. cinaedi and the development of atherosclerosis. However, age (OR: 1.042), total cholesterol (≥200 mg/dL, OR: 1.849), and high-density lipoprotein (≥50 mg/dL, OR: 0.745, as protective) levels have a significant role in the pathogenesis of atherosclerosis development. We also suggest that the presence of H. cinaedi may contribute to the risk of atherosclerosis development due to the univariate comparison result.

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