Diagnosis of pulmonary and extrapulmonary tuberculosis based on detection of mycobacterial antigen 85B by immuno-PCR

Singh, Netrapal; Sreenivas, Vishnubhatla; Gupta, K. B.; Chaudhary, Anil; Mittal, Anshu; Varma-Basil, Mandira; Prasad, Rajendra; Gakhar, Surender K.; Khuller, G. K.; Mehta, Promod K

doi:10.1016/j.diagmicrobio.2015.08.015

Cited by 29 publications

(16 citation statements)

References 21 publications

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“…Similarly, the conventional I-PCR in robostrip wells (solid format) also revealed a sample matrix effect as higher LOD (100-fold) of 100 fg/mL was observed for Ag85B detection in spiked saliva samples. 2 In contrast to synthetic MBs, Wacker et al 30 employed biogenic magnetosome particles isolated from Magnetospirillum gryphiswaldense for designing magneto I-PCR for the detection of recombinant HBsAg in human serum with an LOD of 320 pg/mL, whereas we observed an LOD of 10 fg/mL in human saliva for the detection of recombinant ESAT-6. The use of biogenic magnetosomes has been reported to possess some advantages over the synthetic MBs as better signal-to-noise ratios and lesser SDs are observed owing to their smaller size, monodispersity and higher magnetization.…”

Section: Resultsmentioning

confidence: 68%

“…During the last two decades, an unprecedented interest has been emerged for designing new TB diagnostic tools but still a reliable and accurate test for rapid diagnosis for all forms of TB, ie, pulmonary, extrapulmonary, TB-HIV coinfection and latent TB is lacking. We developed immuno-PCR (I-PCR) assay based on the detection of array of Mycobacterium tuberculosis antigens, ie, Ag85B (Antigen 85B, Rv1886c), ESAT-6 (early secreted antigenic target-6), cord factor (trehalose 6,6′-dimycolate) and PstS1 (phosphate-specific transporter lipoprotein, Rv0934), [2][3][4] which certainly revealed better sensitivity than ELISA. ESAT-6 encoded by region of difference (RD1) is M. tuberculosis complex specific and is considered as a potential biomarker for the diagnosis of TB by ELISA and I-PCR, either alone or in combination with other RD1 and RD2 antigens such as CFP-10 (culture filtrate protein-10, Rv3874) and MPT64 (mycobacterial protein from species tuberculosis-64, Rv1980c).…”

Section: Introductionmentioning

confidence: 99%

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Detection of <em>Mycobacterium tuberculosis</em> purified ESAT-6 (Rv3875) by magnetic bead-coupled gold nanoparticle-based immuno-PCR assay

Singh¹,

Dahiya²,

Radhakrishnan³

et al. 2018

IJN

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PurposeImmuno-PCR (I-PCR), an ultrasensitive method, combines the versatility of ELISA with the exponential amplification capacity of PCR. Coupling of detection antibodies with the reporter DNA is a critical step of I-PCR. Gold nanoparticles (GNPs) and magnetic beads (MBs) are relatively easy to attach with the antibodies and DNA. Therefore, we designed MB-coupled GNP-based I-PCR (MB-GNP-I-PCR) assay for the detection of Mycobacterium tuberculosis antigen.MethodsGNPs were synthesized by chemical reduction and seed-mediated synthesis. Functionalized GNPs were prepared by coupling GNPs with the detection antibodies and reporter DNA and were characterized. Detection limit of M. tuberculosis-specific purified early secreted antigenic target-6 (ESAT-6) (Rv3875) was determined by MB-GNP-I-PCR.ResultsTransmission electron microscopy revealed spherical and slightly polydispersed GNPs of ~20 and ~60 nm size. Coupling of antibodies to GNPs was indicated by a shift in absorption maxima from 524 to 534 nm, which was confirmed by transmission electron microscopy. A color reaction with ELISA and the presence of 76 bp product by PCR further validated the coupling of detection antibodies and signal DNA to the functionalized GNPs. Also, attachment of capture antibodies with MBs was confirmed by magneto-ELISA. Detection limit of purified ESAT-6 by MB-GNP-I-PCR was determined to be 10 fg/mL, 105-fold lower than analogous ELISA. Notably, no sample matrix effect was observed in the saliva samples of healthy individuals spiked with the purified ESAT-6.ConclusionUnlike conventional I-PCR (solid format), MB-GNP-I-PCR (liquid format) is relatively simple with the reduced background signals, which can be further exploited for the clinical diagnosis of tuberculosis.

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Section: Resultsmentioning

confidence: 68%

Section: Introductionmentioning

confidence: 99%

Detection of <em>Mycobacterium tuberculosis</em> purified ESAT-6 (Rv3875) by magnetic bead-coupled gold nanoparticle-based immuno-PCR assay

Singh¹,

Dahiya²,

Radhakrishnan³

et al. 2018

IJN

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“…Picomolar LODs were also obtained in the highly complex matrix of 40% serum (0.07 to 5.38 pM). In 40% serum, SOMAmer 14494-124 had the lowest LOD, 0.07 pM, or ∼5 pg/ml of antigen 85C in serum, which is roughly 3 orders of magnitude below reported LOD values for indirect enzyme-linked immunosorbent assay (ELISA) using anti-antigen 85 antibodies ( 51 , 52 ) but about 3 orders of magnitude above the LOD of antigen 85B detection by immuno-PCR ( 53 ). LODs in urine protein concentrates ranged from 0.06 to 55.47 pM.…”

Section: Discussionmentioning

confidence: 70%

Potential of High-Affinity, Slow Off-Rate Modified Aptamer Reagents for Mycobacterium tuberculosis Proteins as Tools for Infection Models and Diagnostic Applications

et al. 2017

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Direct pathogen detection in blood to diagnose active tuberculosis (TB) has been difficult due to low levels of circulating antigens or due to the lack of specific, high-affinity binding reagents and reliable assays with adequate sensitivity. We sought to determine whether slow off-rate modified aptamer (SOMAmer) reagents with subnanomolar affinity for Mycobacterium tuberculosis proteins (antigens 85A, 85B, 85C, GroES, GroEL2, DnaK, CFP10, KAD, CFP2, RplL, and Tpx) could be useful to diagnose tuberculosis. When incorporated into the multiplexed, array-based proteomic SOMAscan assay, limits of detection reached the subpicomolar range in 40% serum. Binding to native M. tuberculosis proteins was confirmed by using M. tuberculosis culture filtrate proteins and fractions from infected macrophages and via affinity capture assays and subsequent mass spectrometry. Comparison of serum from culture-positive pulmonary TB patients and TB suspects systematically ruled out for TB revealed small but statistically significant (P < 0.0001) differences in the median M. tuberculosis signals and in specific pathogen markers, such as antigen 85B. Samples where many M. tuberculosis aptamers produced high signals were rare exceptions. In concentrated, protein-normalized urine from TB patients and non-TB controls, the CFP10 (EsxB) SOMAmer yielded the most significant differential signals (P < 0.0276), particularly in TB patients with HIV coinfection. In conclusion, direct M. tuberculosis antigen detection proved difficult even with a sensitive method such as SOMAscan, likely due to their very low, subpicomolar abundance. The observed differences between cases and controls had limited diagnostic utility in serum and urine, but further evaluation of M. tuberculosis SOMAmers using other platforms and sample types is warranted.

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“…CFP‐10 (Rv3874), ESAT‐6 (Rv3875), Ag85B (Rv1886c) and cord factor (trehalose‐6,6’‐dimycolate) in TB patients by I‐PCR assay, wherein detection of Ag85B revealed the most promising results (Singh et al . 2015; Mehta et al . 2017).…”

Section: Introductionmentioning

confidence: 99%

Diagnosis of osteoarticular tuberculosis by immuno‐PCR assay based on mycobacterial antigen 85 complex detection

Khan

Singh

Sharma

et al. 2021

Letters Applied Microbiology

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Diagnosis of osteoarticular tuberculosis (OATB) exhibits serious challenges owing to paucibacillary nature of specimens and localization of disease at sites that are difficult to access. We recently developed indirect immuno‐PCR (I‐PCR) and real‐time I‐PCR (RT‐I‐PCR) assays for the detection of mycobacterial antigen 85 complex (Ag85) in OATB patients. Detection limits for the purified Ag85 protein were found to be 1 and 41 fg ml−1 by I‐PCR and RT‐I‐PCR, respectively, which were at least 105‐fold lower than respective ELISA. While spiking synovial fluids of non‐TB control subjects with the purified Ag85 protein, LODs of 100 and 120 fg ml−1 were obtained by I‐PCR and RT‐I‐PCR, respectively, thus demonstrating the sample matrix effect. Sensitivities of 87·5 and 70·5% were observed in bodily fluids of confirmed (n = 8) and clinically suspected (n = 51) OATB cases, respectively, by I‐PCR, with a specificity of 93·9% (n = 33). Markedly, the sensitivities obtained by I‐PCR/RT‐I‐PCR were significantly higher (P < 0·05–0·01) than ELISA and GeneXpert assay (n = 30). However, no substantial difference in sensitivity was observed between the I‐PCR and RT‐I‐PCR assays. After further improving the accuracy of I‐PCR, this test may lead to development of an attractive diagnostic kit.

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Diagnosis of pulmonary and extrapulmonary tuberculosis based on detection of mycobacterial antigen 85B by immuno-PCR

Cited by 29 publications

References 21 publications

Detection of <em>Mycobacterium tuberculosis</em> purified ESAT-6 (Rv3875) by magnetic bead-coupled gold nanoparticle-based immuno-PCR assay

Detection of <em>Mycobacterium tuberculosis</em> purified ESAT-6 (Rv3875) by magnetic bead-coupled gold nanoparticle-based immuno-PCR assay

Potential of High-Affinity, Slow Off-Rate Modified Aptamer Reagents for Mycobacterium tuberculosis Proteins as Tools for Infection Models and Diagnostic Applications

Diagnosis of osteoarticular tuberculosis by immuno‐PCR assay based on mycobacterial antigen 85 complex detection

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