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Evaluation of the sensitivity and specificity of a ligase chain reaction test kit for the detection of Chlamydia trachomatis.

ABSTRACT: The sensitivity and specificity of a newly developed ligase chain reaction (LCR) test kit were examined by the use of highly purified elementary bodies (EBs) and in situ inclusions containing reticulate bodies only. The performance of the LCR kit was compared with a commercially available polymerase chain reaction (PCR) test kit, AMPLICOR Chlamydia trachomatis. The number of EBs and inclusions of C trachomatis, respectively, at the detection limit of both kits were two EBs and one inclusion per assay. Neither kit cross-reacted with C pneumoniae, C psittaci and C pecorum EBs or reticulate bodies.

SUBMITTER: Miyashita N

PROVIDER: S-EPMC500548 | biostudies-other | 1996 Jun

REPOSITORIES: biostudies-other

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Evaluation of the sensitivity and specificity of a ligase chain reaction test kit for the detection of Chlamydia trachomatis.

Miyashita N N Matsumoto A A Niki Y Y Matsushima T T

Journal of clinical pathology 19960601 6

The sensitivity and specificity of a newly developed ligase chain reaction (LCR) test kit were examined by the use of highly purified elementary bodies (EBs) and in situ inclusions containing reticulate bodies only. The performance of the LCR kit was compared with a commercially available polymerase chain reaction (PCR) test kit, AMPLICOR Chlamydia trachomatis. The number of EBs and inclusions of C trachomatis, respectively, at the detection limit of both kits were two EBs and one inclusion per ...[more]

PMID: 8763272

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Project description:UnlabelledProtein phosphorylation has become increasingly recognized for its role in regulating bacterial physiology and virulence. Chlamydia spp. encode two validated Hanks'-type Ser/Thr protein kinases, which typically function with cognate protein phosphatases and appear capable of global protein phosphorylation. Consequently, we sought to identify a Ser/Thr protein phosphatase partner for the chlamydial kinases. CTL0511 from Chlamydia trachomatis L2 434/Bu, which has homologs in all sequenced Chlamydia spp., is a predicted type 2C Ser/Thr protein phosphatase (PP2C). Recombinant maltose-binding protein (MBP)-tagged CTL0511 (rCTL0511) hydrolyzed p-nitrophenyl phosphate (pNPP), a generic phosphatase substrate, in a MnCl2-dependent manner at physiological pH. Assays using phosphopeptide substrates revealed that rCTL0511 can dephosphorylate phosphorylated serine (P-Ser), P-Thr, and P-Tyr residues using either MnCl2 or MgCl2, indicating that metal usage can alter substrate preference. Phosphatase activity was unaffected by PP1, PP2A, and PP3 phosphatase inhibitors, while mutation of conserved PP2C residues significantly inhibited activity. Finally, phosphatase activity was detected in elementary body (EB) and reticulate body (RB) lysates, supporting a role for protein dephosphorylation in chlamydial development. These findings support that CTL0511 is a metal-dependent protein phosphatase with broad substrate specificity, substantiating a reversible phosphorylation network in C. trachomatisImportanceChlamydia spp. are obligate intracellular bacterial pathogens responsible for a variety of diseases in humans and economically important animal species. Our work demonstrates that Chlamydia spp. produce a PP2C capable of dephosphorylating P-Thr, P-Ser, and P-Tyr and that Chlamydia trachomatis EBs and RBs possess phosphatase activity. In conjunction with the chlamydial Hanks'-type kinases Pkn1 and PknD, validation of CTL0511 fulfills the enzymatic requirements for a reversible phosphoprotein network. As protein phosphorylation regulates important cellular processes, including metabolism, differentiation, and virulence, in other bacterial pathogens, these results set the stage for elucidating the role of global protein phosphorylation in chlamydial physiology and virulence.

Dataset Information

Evaluation of the sensitivity and specificity of a ligase chain reaction test kit for the detection of Chlamydia trachomatis.

Publications

Evaluation of the sensitivity and specificity of a ligase chain reaction test kit for the detection of Chlamydia trachomatis.

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