Using genomic DNA copies to enumerate Mycobacterium tuberculosis load in macaque tissue samples

OBJECTIVESIn this study, we sought to evaluate the performance of the Xpert MTB/RIF (Cepheid) assay for the detection of Mycobacterium tuberculosis (MTB) complex DNA on fresh and formalin-fixed, paraffin-embedded (FFPE) tissue specimens from oncology patients in an area with a low prevalence of tuberculosis. We also aimed to retrospectively assess the potential impact of Xpert MTB/RIF on the duration of airborne infection isolation (AII).SETTINGA 473-bed, tertiary-care cancer center in New York City.DESIGNA total of 203 tissue samples (101 FFPE and 102 fresh) were tested using Xpert MTB/RIF, including 133 pulmonary tissue samples (65.5%) and 70 extrapulmonary tissue samples (34.5%). Acid-fast bacilli (AFB) culture was used as the diagnostic gold standard. The limit of detection (LOD) and reproducibility were also evaluated for both samples types using contrived specimens. The potential impact of the Xpert MTB PCR assay on tissue samples from AII patients on AII duration was retrospectively assessed.RESULTSUsing the Xpert MTB/RIF for fresh tissue specimens, the sensitivity was 50% (95% CI, 1.3%–98.7%) and the specificity was 99% (95% CI, 94.5%–99.9%). For FFPE tissue specimens, the sensitivity was 100% (95% CI, 63.1%–100%) and the specificity was 98.3% (95% CI, 95.5%–100%. The LOD was 103 colony-forming units (CFU)/mL for both fresh and FFPE tissue specimens, and the Xpert MTB/RIF was 100% reproducible at concentrations 10 times that of the LOD. With an expected turnaround time of 24 hours, the Xpert MTB PCR could decrease the duration of AII from a median of 8 days to a median of 1 day.CONCLUSIONSThe Xpert MTB/RIF assay offers a valid option for ruling out Mycobacterium tuberculosis complex (MTBC) on tissue samples from oncology patients and for minimizing AII resource utilization.Infect Control Hosp Epidemiol 2018;39:462–466

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Two Novel Nonradioactive Polymerase Chain Reaction-Based Assays of Dried Blood Spots, Genomic DNA, or Whole Cells for Fast, Reliable Detection of Z and S Mutations in the α1-Antitrypsin Gene

Clinical Chemistry ◽

10.1093/clinchem/38.10.2100 ◽

1992 ◽

Vol 38 (10) ◽

pp. 2100-2107 ◽

Cited By ~ 17

Author(s):

B S Andresen ◽

I Knudsen ◽

P K Jensen ◽

K Rasmussen ◽

N Gregersen

Keyword(s):

Polymerase Chain Reaction ◽

Genomic Dna ◽

Dried Blood Spots ◽

Chain Reaction ◽

Tissue Samples ◽

Whole Cells ◽

Blood Spots ◽

Allele Specific ◽

Polymerase Chain ◽

Alpha 1 Antitrypsin

Abstract Two new nonradioactive polymerase chain reaction (PCR)-based assays for the Z and S mutations in the alpha 1-antitrypsin gene are presented. The assays take advantage of PCR-mediated mutagenesis, creating new diagnostic restriction enzyme sites for unambiguous discrimination between test samples from individuals who are normal, heterozygous, or homozygous for the mutations. We show that the two assays can be performed with purified genomic DNA as well as with boiled blood spots. The new assays were validated by parallel testing with a technique in which PCR is combined with allele-specific oligonucleotide (ASO) probes. In all cases tested the results obtained by the different techniques were in accordance. The new assays can be used for prenatal diagnostics and can be performed directly with boiled tissue samples. Because the new assays are easy to perform and reliable, we conclude that they are well suited for routine diagnosis.

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Rapid Detection of Mycobacterium Tuberculosis in Lung Tissue and Analysis of Drug Resistance

Nanoscience and Nanotechnology Letters ◽

10.1166/nnl.2019.2939 ◽

2019 ◽

Vol 11 (5) ◽

pp. 728-735

Author(s):

Junlin Chen ◽

Fei Huang ◽

Feifan Xu ◽

Mei Qu ◽

Delin Gu ◽

...

Keyword(s):

Drug Resistance ◽

Mycobacterium Tuberculosis ◽

Dna Sequencing ◽

Lung Tissue ◽

Culture System ◽

Melting Curve ◽

Gyra Gene ◽

Tissue Samples ◽

Curve Method ◽

Acid Fast Staining

This study investigated the methods for optimizing the workflow for improving the diagnostic efficiency for detection of Mycobacterium tuberculosis (MTB) in lung tissue specimens. A total of 278 specimens were used in this study. M. tuberculosis in fresh lung tissue samples was detected by BACTEC MGIT 960 culture system, culturing L-form MTB, rifampicin (RFP) and levofloxacin (LVFX) susceptibility test, and ribonucleic acid (RNA) simultaneous amplification and testing (SAT). Specimen samples were embedded in paraffin and serially sectioned. The sections were subjected to Ziehl-Neelsen staining and Intensified Kinyoun (IK) acid-fast staining. The suspected MTB or L-form MTB specimens were further investigated by deoxyribonucleic acid (DNA) sequencing and fluorescence polymerase chain reaction (PCR) melting curve method to detect the mutations in rpoB gene and gyrA gene. Thirteen specimens were suspected as MTB positive, and 37 specimens were suspected to be L-form MTB positive by Ziehl-Neelsen staining and IK acid-fast staining. Among the 50 specimens, the number of MTB positive specimens detected by SAT, DNA sequencing, and fluorescence PCR melting curve method was 43, 44, and 45, respectively. Only 11 MTB positive specimens were detected by BACTEC MGIT 960 culture system or by culturing L-form MTB. Mutations detected in rpoB gene and gyrA gene by fluorescence PCR melting curve method were similar to those detected by DNA sequencing. Some specimens, detected by melting curve method, exhibited varied drug resistance to RFP, suggesting heterogeneous resistance. Among the remaining 228 specimens, there was no MTB or L-form MTB detected by BACTEC MGIT 960 culture system. However, 5 specimens were detected to be MTB positive by the SAT method. The fluorescent PCR melting curve method, which has a high level of automation and high sensitivity and specificity, could effectively detect heterozygous drug resistance of MTB in lung tissue samples, which is important for clinicians to effectively formulate a therapeutic strategy.

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Improved protocol for extraction of genomic DNA from formalin-fixed paraffin-embedded tissue samples without the use of xylene

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2016-0108 ◽

2016 ◽

Vol 54 (12) ◽

Cited By ~ 4

Author(s):

Arizumi Kikuchi ◽

Takahiro Sawamura ◽

Osami Daimaru ◽

Masanobu Horie ◽

Kazutoshi Sasaki ◽

...

Keyword(s):

Genomic Dna ◽

Tissue Samples ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Formalin Fixed

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Ribokinase screened from T7 phage displayed Mycobacterium tuberculosis genomic DNA library had good potential for the serodiagnosis of tuberculosis

Applied Microbiology and Biotechnology ◽

10.1007/s00253-019-09756-5 ◽

2019 ◽

Vol 103 (13) ◽

pp. 5259-5267 ◽

Cited By ~ 1

Author(s):

Dan Luo ◽

Li Wang ◽

Haican Liu ◽

Lingling Li ◽

Yating Liao ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Genomic Dna ◽

Dna Library ◽

Genomic Dna Library ◽

Good Potential ◽

T7 Phage

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KRAS mutation analysis in genomic DNA isolated from formalin-fixed paraffin-embedded ovarian tissue: evaluation of a strip-based reverse-hybridisation assay

Journal of Clinical Pathology ◽

10.1136/jcp.2010.081414 ◽

2011 ◽

Vol 64 (3) ◽

pp. 252-256 ◽

Cited By ~ 6

Author(s):

Gernot Kriegshäuser ◽

Veronika Auner ◽

Eva Schuster ◽

Barbara Holzer ◽

Christian Oberkanins ◽

...

Keyword(s):

Genomic Dna ◽

Ovarian Tissue ◽

Analytical Sensitivity ◽

Tissue Samples ◽

Formalin Fixed Paraffin ◽

Ffpe Samples ◽

Formalin Fixed Paraffin Embedded ◽

Sensitivity Specificity ◽

Formalin Fixed ◽

Reverse Hybridisation Assay

AimsTo evaluate a reverse-hybridisation assay (strip assay) designed for the sensitive detection of 10 mutations in codons 12 and 13 of the KRAS gene. The strip assay relies on mutant-enriched PCR followed by reverse-hybridisation of biotinylated amplification products to oligonucleotide probes immobilised as an array of parallel lines on nitrocellulose test strips.MethodsThe strip assay was used to analyse genomic DNA isolated from 120 formalin-fixed paraffin-embedded (FFPE) ovarian tissue samples. The samples were analysed in parallel using a biochip-based protocol (biochip assay) covering the same mutation spectrum, and results were compared with respect to sensitivity, specificity and operational input.ResultsThe strip assay identified 19 (16%) of 120 FFPE samples to carry a KRAS mutation; results were in agreement with those obtained by biochip hybridisation. Both assays had an analytical sensitivity of 1% when performed on FFPE-extracted DNA with approximately the same operational input needed for post-PCR processing. In contrast to the biochip assay, strip assay hybridisation may be automated to a large extent.ConclusionsThe strip assay is an accurate and sensitive tool for the low to medium throughput detection of KRAS mutation in genomic DNA isolated from FFPE tissue.

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Amorphus globosus foetuses in Polish Holstein cattle: anatomical, histological, and genetic studies

Journal of Veterinary Research ◽

10.2478/jvetres-2019-0057 ◽

2019 ◽

Vol 63 (3) ◽

pp. 391-398

Author(s):

Marek Gehrke ◽

Beata Blaszak ◽

Monika Stachowiak ◽

Izabela Szczerbal ◽

Barbara Stefańska ◽

...

Keyword(s):

Genomic Dna ◽

Histological Analysis ◽

Holstein Cattle ◽

Multiple Pregnancies ◽

Comprehensive Description ◽

Blood Leukocytes ◽

Genetic Studies ◽

Tissue Samples ◽

Linked Gene ◽

Healthy Siblings

Abstract Introduction A comprehensive description is presented of four novel cases ofamorphus globosus (ag) foetuses originating from multiple pregnancies of Polish Holstein cows. Material and Methods Four amorphic foetuses were delivered by three cows. Tissue samples were collected during autopsy, embedded in paraffin, sectioned, and stained with haematoxylin and eosin. Genomic DNA was isolated from tissue samples of abnormal foetuses and from blood leukocytes of their healthy siblings. PCR reactions were used to reveal the presence of Y-linked genes (SRY and AMELY) and an X-linked gene (AMELX). Results All foetuses were classified to the groupholoacardius amorphous (anideus). Molecular analysis clearly showed that at 17 microsatellite loci, the studied amorphous foetuses had identical genotypes to the viable co-twins. Conclusion Foetuses had monozygotic origin. Histological analysis showed a low level of development of tissues of meso- and ectodermal origin, as well as features of degrading patterns.

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Two target genes based multiple cross displacement amplification combined with a lateral flow biosensor for the detection of Mycobacterium tuberculosis complex

BMC Microbiology ◽

10.1186/s12866-021-02328-6 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Junfei Huang ◽

Ziyu Xiao ◽

Xinggui Yang ◽

Xu Chen ◽

Xiaojuan Wang ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Real Time ◽

Genomic Dna ◽

Target Genes ◽

Limit Of Detection ◽

Lateral Flow ◽

Product Analysis ◽

Prevention And Treatment ◽

Multiple Cross ◽

Colorimetric Indicator

Abstract Background Tuberculosis (TB) is a serious chronic infectious disease caused by Mycobacterium tuberculosis complex (MTBC). Hence, the development of a novel, simple, rapid and sensitive method to detect MTBC is of great significance for the prevention and treatment of TB. Results In this study, multiple cross displacement amplification (MCDA) combined with a nanoparticle-based lateral flow biosensor (LFB) was developed to simultaneously detect two target genes (IS6110 and mpb64) of MTBC (MCDA-LFB). One suite of specific MCDA primers designed for the IS6110 and mpb64 genes was validated using genomic DNA extracted from the reference strain H37Rv. The MCDA amplicons were analyzed using a real-time turbidimeter, colorimetric indicator (malachite green, MG) and LFBs. The optimal amplification temperature and time were confirmed, and the MCDA-LFB method established in the current report was evaluated by detecting various pathogens (i.e., reference strains, isolates and clinical sputum samples). The results showed that the two sets of MCDA primers targeting the IS6110 and mpb64 genes could effectively detect MTBC strains. The optimal reaction conditions for the MCDA assay were determined to be 67 °C for 35 min. The MCDA assay limit of detection (LoD) was 100 fg per reaction for pure genomic DNA. The specificity of the MCDA-LFB assay was 100%, and there were no cross-reactions for non-MTBC strains. For sputum samples and MTBC strain detection, the positive rate of MCDA-LFB for the detection of MTBC strains was consistent with seminested automatic real-time PCR (Xpert MTB/RIF) and higher than acid-fast staining (AFS) and culture assays when used for sputum samples. The MCDA-LFB assay was a rapid tool, and the whole procedure for MCDA-LFB, including DNA template preparation, MCDA reaction and amplification product analysis, was completed within 70 min. Conclusion The MCDA-LFB assay targeting the IS6110 and mpb64 genes is a simple, rapid, sensitive and reliable detection method, and it has potential significance for the prevention and treatment of TB.

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