Immuno-PCR, a new technique for the serodiagnosis of tuberculosis

Mehta, Promod K; Dahiya, Bhawna; Sharma, Sunil; Singh, Netrapal; Dharra, Renu; Thakur, Zoozeal; Mehta, Neeru; Gupta, K. B.; Gupta, Mahesh; Chaudhary, Dhruva

doi:10.1016/j.mimet.2017.05.009

Cited by 40 publications

(27 citation statements)

References 81 publications

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“…5,6 In fact, ESAT-6 is an immunodominant T-cell stimulatory antigen recognized by specific interferon-γ secreting T cells, which are present in higher numbers in TB patients with active infection than in uninfected individuals. 7 Strikingly, I-PCR could detect a significantly higher number of samples from smear-negative pulmonary and paucibacillary extrapulmonary TB patients 3,8 and thus showed superiority in sensitivity over the routine diagnostic methods, ie, smear, histopathology and nucleic acid amplification tests such as PCR. Coupling of detection antibodies with the reporter DNA is a crucial step of I-PCR, and various strategies are employed such as streptavidin-protein A, streptavidin-biotin setup, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1carboxylate (SMCC) system and recombinant phage particle where surface displayed single-chain variable fragments and phage DNAs themselves act as detection antibodies and DNA tag, respectively, in phage display-mediated I-PCR.…”

Section: Introductionmentioning

confidence: 99%

“…Coupling of detection antibodies with the reporter DNA is a crucial step of I-PCR, and various strategies are employed such as streptavidin-protein A, streptavidin-biotin setup, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1carboxylate (SMCC) system and recombinant phage particle where surface displayed single-chain variable fragments and phage DNAs themselves act as detection antibodies and DNA tag, respectively, in phage display-mediated I-PCR. [8][9][10] Over the last two decades, nanobiotechnology has emerged as the most promising tool for clinical diagnostics. 11 In particular, due to small size (1-100 nm), corresponding high surface-to-volume ratio and unusual target binding properties, nanoparticles (NPs) have been used as signal amplification tools.…”

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: Introductionmentioning

confidence: 99%

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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“…Although ELISA can detect nonnucleic acid molecules, it fails when the target antigen concentration is low. Contrarily, Immuno-PCR (I-PCR) assay can detect ultralow amount of target antigens [17]. In fact, I-PCR comprises the versatility of ELISA with the enormous amplification capacity of PCR, thereby exhibiting many-fold higher sensitivity than ELISA [17].…”

Section: Immuno-pcrmentioning

confidence: 99%

“…Contrarily, Immuno-PCR (I-PCR) assay can detect ultralow amount of target antigens [17]. In fact, I-PCR comprises the versatility of ELISA with the enormous amplification capacity of PCR, thereby exhibiting many-fold higher sensitivity than ELISA [17]. Moreover, urine is quite easy to obtain that has a lesser risk of infection to the laboratory workers but it carries low concentration of M. tuberculosis biomarkers [18].…”

Section: Immuno-pcrmentioning

confidence: 99%

“…Moreover, urine is quite easy to obtain that has a lesser risk of infection to the laboratory workers but it carries low concentration of M. tuberculosis biomarkers [18]. Detection of mycobacterial lipoarabinomannan (LAM) within urine samples is considered as an attractive biomarker, which has been demonstrated in HIV-coinfected and HIVuninfected TB individuals by lateral flow immunochromatography and ELISA methods though poor sensitivities were observed in HIV-uninfected TB individuals [17,19]. However, urinary extracellular vesicles (EVs) isolated from TB patients can furnish intact pathogen-derived biomarkers and also concentrate those biomarkers [19].…”

Section: Immuno-pcrmentioning

confidence: 99%

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Recent Trends in Diagnosis of Urogenital Tuberculosis

Mehta

Kamra

2020

Future Microbiol.

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the efficient diagnosis of UGTB by reliable tests would certainly reduce the infertility in both men and women and the other sequelae associated with disease. " Urogenital tuberculosis (UGTB) is the second most common form of extrapulmonary TB (EPTB) in developing nations with severe epidemic situations. For example, India (comprising 45% of EPTB cases) and the third most common form in locations with a lower incidence of TB [1,2], which include both renal and genital TB. However, concomitant UGTB and pulmonary TB (PTB) are found in 2-10 and 15-20% of patients in developed and developing nations, respectively [2]. In fact, UGTB occurs after a prolonged period of latent infection followed by hematogenous spread to the kidneys, fallopian tubes and epididymis [3]. The clinical manifestations of female UGTB include primary or secondary infertility, menstrual dysfunction and pelvic inflammatory disease [4]. Infertility is the most common complication, which occurs in 5-16% of Indian women caused by irreversible damage to the fallopian tubes. Mostly, fallopian tubes are associated with congestion and miliary tubercles, while endometrium is linked with caseation and ulceration in 50-80% cases, resulting in Asherman's syndrome [5]. Similarly, the most frequent sites for male UGTB are the epididymis and prostate followed by seminal vesicles and testicles originating from renal foci, which also lead to infertility [6]. Owing to asymptomatic nature and varied clinical presentations (ranging from vague urinary symptoms to chronic kidney disease) that mimic several urologic and genital diseases, diagnosis of UGTB is a daunting challenge [3].Smear/culture, histopathology, interferon-γ release assays (IGRAs), intravenous pyelography, laparoscopy and nucleic acid amplification tests (NAATs) are the main modalities employed in the diagnosis of UGTB [6][7][8], which have certain limitations. Smear microscopy and culture for Mycobacterium tuberculosis identification (on Lowenstein-Jensen medium or BACTEC-460/MGIT-960) are commonly used methods but they lead to poor sensitivities due to paucibacillary nature of specimens [7]. Though urine culture is considered the gold standard, it requires skillful technicians and has a prolonged turnaround time of approximately 6-8 weeks [7] when irreparable damage to the fallopian tubes and epididymis already occurs. Delayed diagnosis also leads to nonfunctioning unilateral kidney, contracted bladder and renal failure [1]. Endoscopic procedures such as laparoscopy/hysteroscopy are often linked with operative risks and mostly cause flaring of infection [5], while histopathological examination can be confirmatory only when it demonstrates acid-fast bacilli in the tissues that are very scarce in endometrial biopsies (EBs) [7]. Intravenous pyelography may be suggestive of UGTB but it is nonspecific and often mimics other pathologic lesions [1]. IGRAsImmunological procedures such as IGRAs are widely employed for the early detection of UGTB cases. Notably, Kim et al. [8] reported a sensitivity of 52.6%...

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