Rapid Identification of <i>Klebsiella pneumoniae</i>, <i>Corynebacterium kutscheri</i>, and <i>Streptococcus pneumoniae</i> Using Triplex Polymerase Chain Reaction in Rodents

Abstract

Klebsiella pneumoniae, Corynebacterium kutscheri, and Streptococcus pneumoniae are important pathogens that cause respiratory infections in laboratory rodents. In this study, we used species-specific triplex PCR analysis to directly detect three common bacterial pathogens associated with respiratory diseases. Specific targets were amplified with conventional PCR using the tyrB gene from K. pneumoniae, gyrB gene from C. kutscheri, and ply gene from S. pneumoniae. Our primers were tested against purified DNA from another eleven murine bacteria to determine primer specificity. Under optimal PCR conditions, the triplex assay simultaneously yielded a 931 bp product from K. pneumoniae, a 540 bp product from C. kutscheri, and a 354 bp product from S. pneumoniae. The triplex assay detection thresholds for pure cultures were 10 pg for K. pneumoniae and S. pneumoniae, and 100 pg for C. kutscheri. All three bacteria were successfully identified in the trachea and lung of experimentally infected mice at the same time. Our triplex PCR method can be used as a useful method for detecting pathogenic bacterial infections in laboratory rodents.

Development of a bead-based suspension array for the detection of pathogens in acute respiratory tract infections

“…Six kinds of primers and probes corresponding to sequences from the SARS coronavirus, the Influenza A virus, the highly pathogenic avian influenza A H5N1, the human metapneumovirus, and the human bocavirus were designed based on previous studies 6,15,[17][18][19][20][21] and optimized for our research purposes. Fourteen pathogen sequences, including human cytomegalovirus transmembrane protein gene (X04650), human adenovirus type 6 hexon protein gene (DQ149613), human parainfluenza virus 1 HN gene (U70942), human parainfluenza virus 2 HN gene (AB367954), human parainfluenza virus 3 HN gene (AB623457), human respiratory syncytial virus nucleocapsid gene (X00001), Mycoplasma pneumoniae P1 gene (KF154759), Chlamydia pneumoniae rpoB gene (KC305894), Staphylococcus aureus tuf gene (HM352930), Streptococcus pneumoniae ply gene (GU968401), Klebsiella pneumoniae phoE gene (AF009172), Acinetobacter baumannii Oxa gene (JQ342838), Pseudomonas aeruginosa gryB gene (FJ652722), and Stenotrophomonas maltophilia 23S rRNA gene (HE798556), were retrieved from GenBank and aligned with pathogen sequences from patient samples using the Cluster Omega software in order to confirm pairwise identities.…”

“…Some primer sequences were obtained from literature, 6,[13][14][15]17,22,23 and optimized to meet our research needs. Primers and probes were designed using Primer Premier 5.0 software and synthesized by Invitrogen Trading (Shanghai) Co., Ltd, Shanghai, China.…”

Biology and Diseases of Rats

“…Latent infections may be triggered to become clinical by a variety of stressors that can cause immunosuppression in the host. Definitive diagnosis is accomplished by bacteriologic culture (Fox et al, 1987) or PCR (Jeong et al, 2013). As with other persistent infections, such as mycoplasmosis, disease is more frequent in older animals.…”

The ZKIR Assay, a novel Real-Time PCR Method for the Detection of Klebsiella pneumoniae and Closely Related Species in Environmental Samples

“…Moreover, Kp culture methods have not been validated so far for food safety screening. Some molecular methods (without need of sequencing) have been proposed over the years for the rapid detection of Kp (21–25). They target the 16S-23S rRNA internal transcribed spacer sequence (ITS) (21), coding sequences of tyrB (25), khe (24, 26), chromosomal beta-lactamase ( bla ) genes (27, 28) or other molecular targets (22).…”

“…Some molecular methods (without need of sequencing) have been proposed over the years for the rapid detection of Kp (21–25). They target the 16S-23S rRNA internal transcribed spacer sequence (ITS) (21), coding sequences of tyrB (25), khe (24, 26), chromosomal beta-lactamase ( bla ) genes (27, 28) or other molecular targets (22). Some of these targets are described as able to detect the Kp complex (21), while others were designed for specific members of the Kp complex, such as Kp1 and Kp3 (22, 23).…”