Development of an indirect sandwich ELISA for detection of urinary antigen, using Legionella pneumophila PAL protein

Legionella pneumophila peptidoglycan-associated lipoprotein (PAL) protein is an extremely conserved antigen among Legionella species. In this study, rabbit and rat anti-PAL immunoglobulin G antibodies were produced by immunization with purified, recombinant PAL (r-PAL) protein of L. pneumophila serogroup 1 and used as capture and detection antibodies in the PAL antigen-based enzyme-linked immunosorbent assay (ELISA) to detect urinary PAL antigen. Urine samples were obtained from rats experimentally infected with L. pneumophila serogroup 1. The PAL antigen was measured in urine samples of 40 infected and 40 uninfected rats. After choosing the cut-off value of 0.192, the sensitivity and specificity of the PAL antigen-based ELISA were 87.5 and 97.5 %, respectively. The results obtained by PAL antigen base ELISA were compared with those obtained by Biotest. The PAL antigen was detected efficiently by both of the assays and all of the control human urine samples were negative by the ELISA test. The PAL antigen-based ELISA assay was relatively simple to perform, precise, highly sensitive and specific, and reproducible. Based on our data the PAL antigen-based ELISA described here is the first indirect sandwich ELISA for urinary antigen detection which could easily be applied for diagnosis of Legionnaires disease

Cloning and periplasmic expression of peptidoglycan-associated lipoprotein (PAL) protein of Legionella pneumophila in Escherichia coli

Abstract Introduction and objective: Legionella pneumophila, the etiological agent of Legionnaires’ disease, is an important cause of both community-acquired and nosocomial pneumonia; therefore, rapid diagnosis and early antibiotic treatment of pneumonia are required. Urinary antigen testing to detect Legionella antigen has proven to be the most powerful diagnostic method. Peptidoglycan-associated lipoprotein (PAL) protein of L. pneumophila, as a component of Legionella antigens, will be detected efficiently by the PAL antigen capture assay and is considered as useful diagnostic antigen to diagnose Legionella infection. Because of the transfer of protein to the periplasmic region of Escherichia coli has numerous advantages including separation from cytoplasmic proteins and the concentration of recombinant proteins in periplasm, the aim of this study was to produce periplasmic PAL protein of L. pneumophila in E. coli. Materials and methods: The pal gene of L. pneumophila serogroup 1 was amplified with specific primers, cloned and expressed under pelB signal sequence and T7 lac promoter in pET26b+ plasmid. Results: The cloning was confirmed with digestion and sequencing of recombinant pET- 26b-pal plasmid. The expression of r-PAL protein in cytoplasm and periplasmic space of E. coli was approved by SDS-PAGE and western blotting. Conclusion: The results of this study demonstrated that the r-PAL protein successfully expressed in E. coli

Detection of Helicobacter pylori in Drinking Water by Loop-Mediated Isothermal Amplification

Background: There should be a public environmental reservoir for Helicobacter pylori in the developing countries, such as Iran, due to their high infection rate of over 70%. Epidemiological findings revealed that water could be a possible source of H. pylori transmission. However, high prevalence of H. pylori in drinking water in Kermanshah, West of Iran, was detected in the authors' previously published study. The current study aims at designing a more accurate and rapid procedure to investigate the prevalence of Helicobacter species and cagA gene in drinking water samples in Kermanshah, from October to December 2012. Methods: In the current study, 60 tap water samples were obtained and specific polymerase chain reaction (PCR) targeted cagA and 16s rRNA was performed. A loop-mediated isothermal amplification (LAMP) targeted ureC gene was developed to accurately detect H. pylori in water samples. Results: The prevalence of ureC by PCR, ureC by LAMP and 16s rRNA by PCR were 26.67%, 38%, and 61.67%, respectively. Among 24 samples (40%), 1 of the 2 tests was positive. The prevalence of cagA gene among ureC positive, 16s rRNA positive and all samples were 18.75%, 13.51%, and 10%, respectively. Conclusions: Helicobacter pylori contamination in drinking water was considerably higher using LAMP compared with PCR. It is noteworthy that some H. pylori positive samples were also positive for Caga

Cloning of the Recombinant Cytochrome P450 Cyp141 Protein of Mycobacterium tuberculosis as a Diagnostic Target and Vaccine Candidate

Background: Tuberculosis has been announced as a global emergency by World Health Organization and the second infectious agent of mortality worldwide. The general policy in the development of new vaccines is to develop some vaccines with higher efficiency not only for infants but also for adults compared with the Bacillus Calmette-Guerin vaccine. Recently, cytochrome P450 cyp141 has been introduced as a new target for detecting Mycobacterium tuberculosis from clinical samples. Objectives: The aim of this study was to clone this gene in order to pave the way for more evaluation. Materials and Methods: M. tuberculosis H37Rv DNA was extracted by a standard phenol-chlorophorm protocol. After designing the specific primers, P450 cyp141 gene was replicated by PCR. The purified PCR products were then subcloned into the pTZ57R/T plasmid vector. After extraction, enzyme digestion, and recombinant pTZ57R/T-cyp141 plasmid vector sequencing, the aforementioned products were cloned into a pET-26b plasmid vector. Then, the recombinant pET26b-cyp141 plasmid molecules were transformed to Escherichia coli strain BL21 (DE3) using the transformation method. Next, the recombinant pET26b-cyp141 plasmids were purified and evaluated by the enzyme digestion analysis. Results: The cloning of P450 cyp141 gene was confirmed by the enzyme digestion and sequencing of the recombinant pTZ57R/T-cyp141 and pET26b-cyp141 plasmid vectors. Conclusions: The results of this study demonstrated that the P450 cyp141 gene was successfully cloned into a pET26b plasmid vector as an expression vector. In this paper, for the first time in Iran, this gene was cloned for more purposes, including the expression and purification of the recombinant cytochrome P450 cyp141 protein

Periplasmic expression and one-step purification of urease subunit B of Helicobacter pylori

UreB is one of the urease subunits of Helicobacter pylori and can be used as an excellent antigen candidate for H. pylori vaccination. Easy access to highly purified UreB protein, facilitate advances in therapeutic or preventive strategies. To achieve a simplified purification procedure, the present report represents a novel method of producing recombinant urease subunit B extracellularly. ureB gene from 26,695 standard strain was amplified by PCR and cloned into pET-26b(+) expression vector. UreB was expressed as a soluble, N-terminal pelB and C-terminal hexahistidine-tagged fusion protein (UreB-6His) and secreted into the periplasmic space of Escherichia coli. Expression of the recombinant UreB in E. coli BL21 (DE3) was induced by isopropylthio-β-d-galactoside (IPTG). Expression of UreB was confirmed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and western blot analysis using anti-His monoclonal antibody. UreB-6His protein was extracted from the periplasm by osmotic shock treatment and was purified in one step by Nickel affinity chromatography. In conclusion, the present protocol is easier to perform; more time effective and low cost than earlier methods. Keywords Helicobacter pylori –Urease subunit B–Cloning–Periplasmic expression Periplasmic expression and one-step purification of urease subunit B of Helicobacter pylori (PDF Download Available). Available from: https://www.researchgate.net/publication/227181089_Periplasmic_expression_and_one-step_purification_of_urease_subunit_B_of_Helicobacter_pylori [accessed Dec 09 2017]

Expression and Purification of the Recombinant Cytochrome P450 CYP141 Protein of Mycobacterium Tuberculosis as a Diagnostic Tool and Vaccine Production

Background: Tuberculosis (TB) is regarded as a health problem worldwide, particularly in developing countries. Mycobacterium tuberculosis (M. tuberculosis) is the cause of this disease. Approximately two billion people worldwide are infected by M. tuberculosis and annually about two million individuals die in consequence. Forty million people are estimated to die because of M. tuberculosis over the next 25 years if the measures for controlling this infection are not extensively developed. In the vaccination field, Bacillus Calmette-Guerin (BCG) is still the most effective vaccine but it shows no efficacy in adult pulmonary patients. One of the other problems regarding TB is its appropriate diagnosis. Objectives: In this experimental study, the recombinant cytochrome P450 CYP141 protein of M. tuberculosis was expressed and purified to be used as a vaccine candidate and diagnostic purpose in subsequent investigations. Materials and Methods: The optimization of the cytochrome P450 CYP141 protein expression was evaluated in different conditions. Then, this protein was purified with a resin column of nickel-nitrilotriacetic acid and investigated via Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Western Blotting. Results: The highest expression of the cytochrome P450 CYP141 protein was obtained by the addition of 1 mM of isopropyl beta-D-1-thiogalactopyranoside (IPTG) to the bacterial culture grown to an optical density at 600 nm (OD600) of 0.6, 16 hours after induction. This protein was subsequently purified with a purification of higher than 80%. The results of Western Blotting indicated that the purified protein was specifically detected. Conclusions: In this experimental study, for the first time in Iran the expression and purification of this recombinant protein was done successfully. This recombinant protein could be used as a vaccine candidate and diagnostic purpose in subsequent investigations

Optimization of gene expression and purification of Legionella pneumophila peptidoglycan associated lipoprotein recombinant protein

زمینه و هدف: اکثر تست های موجود برای تشخیص پنومونی لژیونلایی، با مشکلات زیادی از جمله حساسیت و ویژگی پائین و عدم توانایی در فراهم نمودن نتیجه در یک زمان کلینیکی مناسب مواجه هستند. از آنجایی که لیپوپروتئین مرتبط با پپتیدوگلیکان ( PAL ) لژیونلا پنوموفیلا در درون ادرار ترشح شده و به عنوان یک آنتی ژن در تمام سویه های آن وجود دارد. لذا برای تشخیص بیماری لژیونر از طریق ادرار مورد توجه قرار گرفته است. هدف از این مطالعه بهینه سازی بیان و تخلیص پروتئین نوترکیب PAL باکتری لژیونلا پنوموفیلا بوده است. روش بررسی: در این مطالعه تجربی آزمایشگاهی بیان پروتئین PAL با تغییر در پارامترهای دانسیته سلولی، مدت زمان القاء، دمای رشد، غلظت ایزوپروپیل بتا دی تیوگالاکتوپیرانوزید ( IPTG ) و نوع محیط کشت مورد بررسی قرار گرفت. پس از کلونینگ، عصاره پری پلاسمی تهیه و پروتئین نوترکیب PAL با استفاده از ستون رزین نیکل نیتروتری استیک اسید ( NTA ) تخلیص گردید. در نهایت پروتئین نوترکیب PAL با آزمایش وسترن بلاتینگ بررسی شد. یافته ها: با استفاده از محیط کشت Terrific Broth ، شروع القاء در جذب نوری 6/0 (طول موج 600 نانومتر)، غلظت یک میلی مولار ماده القاء کننده IPTG ، دمای رشد 25 درجه سانتیگراد و مدت زمان 15 ساعت پس از القاء، بهترین بیان پروتئین PAL بدست آمد. پروتئین پری پلاسمی نوترکیب PAL با استفاده از ستون رزین NTA با خلوص بیش از 80 تخلیص گردید. نتایج آزمایش وسترن بلاتینگ نیز نشان داد که پروتئین نوترکیب PAL تخلیص شده به طور اختصاصی با آنتی بادی anti-His6-peroxidase شناسایی گردید. نتیجه گیری: با تخلیص پروتئین PAL به میزان بیش از 80 می توان به منظور بررسی پتانسیل آن در تشخیص بیماری لژیونر و تهیه کیت تشخیصی بر اساس الایزا از آن استفاده نمود

Diagnostic accuracy of gold nanoparticle combined with molecular method for detection of Mycobacterium tuberculosis: A systematic review and meta-analysis study

The importance of rapid detection of Mycobacterium tuberculosis (MTB) has become greater than ever. Among biosensors as a rapid test, gold nanoparticles (GNPs) can play an important role in accelerating the diagnosis of TB. This systematic review and meta-analysis study were conducted to evaluate the diagnostic accuracy of GNPs for the diagnosis of MTB. All cross sectional and case control studies as original article about rapid detection of MTB using GNPs combined with molecular methods published in PubMed, Web of Science, and Scopus databases were selected, retrieved and appraised up to September 2021. The sensitivity and specificity of GNPs in different studies ranges from 83.7% to 100% for sensitivity and from 86.6% to 100% for specificity. The highest sensitivity and specificity of GNPs combined with molecular methods was related to the LAMP method. Therefore, GNPs are a simpler, low cost and more efficient way in the clinical diagnosis of TB

Molecular Identification of Mutations Associated with Pyrazinamide-Resistance in Multidrug-Resistant Tuberculosis in Eight Provinces of Iran

Introduction: Multidrug-Resistant Tuberculosis (MDR-TB) is a growing concern which has always played a role in controlling infectious diseases. Pyrazinamide (PZA) is one of the four front line cures of tuberculosis during the first two months of the treatment course. Diagnosis of PZA resistance is imperative in order to optimize the efficacy of new treatment regimens and minimize the risk of developing resistance to new drugs. In addition, high prevalence of PZA resistance, especially among those afflicted with MDR-TB, points to the need for developing those medicinal regimens that can be used in patients with PZA resistant MDR-TB. Aim: The aim of this study was to determine anti-tuberculosis drug resistance rate and identify the correlation between MDRTB and of mutations in pncA gene among Mycobacterium tuberculosis isolates and frequency of mutations associated with PZA resistance in all the isolates. Materials and Methods: This is a cross-sectional descriptive study conducted from April 2014 to June 2015. A total of 118 Mycobacterium tuberculosis (M. tuberculosis) clinical strains were isolates from patients referred to TB reference laboratory of Kermanshah from 8 provinces including: Kermanshah, Lorestan, Hamadan, Ilam, Kurdistan, Ardabil, Uromia and Tabriz. Antimicrobial susceptibility testing was performed using the proportional method and Minimum Inhibitory Concentration (MIC) and mutations in pncA for the PZA resistant isolates were studied using monoplex- Polymerase Chain Reaction (PCR) and then PCR products were sent for sequencing. Results: Among the 118 clinical samples of M. tuberculosis investigated in various parts of Iran, 10 isolates (8.5%) were resistant to Isoniazid, 10 isolates (8.5%) were resistant to Rifampin, 7 isolates (6%) were MDR, and 23 isolates (19.5%) were resistant to PZA. Only did one MDR-TB isolate resistant (14.3%) to PZA show inactive mutation at Glu-122 codon that was found in pncA gene. According to our results, a significant correlation was found between MDR strains and of mutations in pncA gene (pv=0.049). pncA gene was not isolated from any of the PZA resistant isolates. Conclusion: Our findings indicated that only one MDR-TB isolate resistant to PZA showed a mutation in the pncA gene (14.3%) and mutations were not observed in the other PZAresistance isolates. The reason for resistance to PZA in the other PZA-resistance strains might be related to mutations in other genes or to some other factors. Thus, these reasons need to be further investigated in our study population

Prevalence of Mycobacterium tuberculosis mutations associated with isoniazid and rifampicin resistance: A systematic review and meta-analysis

Tuberculosis (TB) is still one of the leading causes of worldwide death, especially following the emergence of strains resistant to isoniazid (INH) and rifampicin (RIF). This study aimed to systematically review published articles focusing on the prevalence of INH and/or RIF resistance-associated mutations of Mycobacterium tuberculosis isolates in recent years. Literature databases were searched using appropriate keywords. The data of the included studies were extracted and used for a random-effects model meta-analysis. Of the initial 1442 studies, 29 were finally eligible to be included in the review.The overall resistance to INH and RIF was about 17.2% and 7.3%, respectively. There was no difference between the frequency of INH and RIF resistance using different phenotypic or genotypic methods. The INH and/or RIF resistance was higher in Asia. The S315T mutation in KatG (23.7 %), C-15 T in InhA (10.7 %), and S531L in RpoB (13.5 %) were the most prevalent mutations. Altogether, the results showed that due to S531L in RpoB, S315T in KatG, and C-15 T in InhA mutations INH- and RIF-resistant M. tuberculosis isolates were widely distributed. Thus, it would be diagnostically and epidemiologically beneficial to track these gene mutations among resistant isolates