Sensitivity and specificity of the colloidal gold assay for M. pneumoniae

To evaluate the specificity of the colloidal gold assay, 105 copies/ml of M. pneumoniae, Staphylococcus aureus, Escherichia coli, Streptococcus pneumonia, Klebsiella pneumoniae, Legionella pneumophila, Haemophilus influenzae, Pseudomonas aeruginosa, anaerobic bacteria, Chlamydia trachomatis, Chlamydia pneumoniae, adenovirus, respiratory syncytial virus, parainfluenza virus, influenza virus, human cytomegalovirus, human metapneumovirus and enteroviruses were assessed in 3 independent experiments. Our results showed that M. pneumoniae was identified correctly by the colloidal gold assay with no cross-reactions found between M. pneumoniae and other pathogens (Data not shown).

To assess the sensitivity of the colloidal gold assay, standard M. pneumoniae was quantified by real-time PCR and submitted to serial 10-fold dilutions to obtain 101 to 107 copies/ml. Different concentrations of standard M. pneumoniae were tested by the colloidal gold assay. As shown in Fig. 3, M. pneumoniae with concentrations of 103–107copies/ml were positive in the colloidal gold assay (3 independent experiments).M. pneumoniae was not detected at 101–102 copies/ml.

Figure 3

Sensitivity of the colloidal gold assay.

Clinical specimens from children with pneumonia

From February to August 2014, 302 children were enrolled in this study. The inclusion criteria were: (1) age < 14 years; (2) patient visiting the Children’s Hospital of Zhejiang, University School of Medicine; (3) primary diagnosis as pneumonia according to known guidelines21. During six months, 302 specimens were collected, including 78 throat swabs and 224 sputum samples in our hospital. Each specimen was mixed with 1.5 ml saline and stored at −70 °C; 1 ml of the mixture was used for real-time PCR and 0.5 ml in the colloidal gold assay. The 302 patients (125 females and 177 males) were 3 months to 10 years old.

The study was performed in accordance with the Declaration of Helsinki and approved by the Medical Ethics Committee of Zhejiang University School of Medicine. All patients provided informed consent.

Preparation of the colloidal gold plate

As shown in Fig. 1, the colloidal gold plate contained three parts: 1) sample well; 2) reagent region; 3) chromatography region. The reagent region contained monoclonal antibody A (mouse) labeled with colloidal gold which targets the M. pneumoniae P1 antigen. The chromatography region included M. pneumoniaemonoclonal antibody B (mouse) which is bound to the Test line position and mouse IgG polyclonal antibody bound to the Control line position in the chromatography. A sample with M. pneumoniae would result in monoclonal antibody A binding with M. pneumoniae and detected by monoclonal antibody B as well as mouse IgG polyclonal antibody (both of Test and Control lines positive). In a sample without M. pneumoniae, only monoclonal antibody A can be detected by IgG polyclonal antibody (Test line negative and Control line positive). The plate was manufactured by Genesis Biodetection & Biocontrol Ltd, Hangzhou, China.

Real-time PCR for M. pneumoniae detection

For real-time PCR, 1 ml of the mixture was transferred into a 1.5-ml microcentrifuge tube aseptically and centrifuged for 5 min at 12000 rpm/min. The cell pellets were resuspended in 50 μl lysis buffer (Da An Gene Co., Ltd., China); 4 μl lysate served as template in real-time PCR amplification based on the TaqMan probe PCR kit (Da An Gene Co., Ltd., China) as reported previously3,22. For each assay, negative and positive quality controls, and four positive quantity plasmid controls (104, 105, 106, and 107 copies/ml) were assessed. Ct values of the four quantity controls were then subjected to log-linear analysis to generate a standard curve used to determine the concentrations of clinical M. pneumoniae samples. Real-time PCR was carried out on an ABI 7500 instrument for 3 min at 95 °C, followed by 40 two-step cycles (15 s at 95 °C and 45 s at 55 °C).

Colloidal gold assay for M. pneumoniae detection

To perform the immune-chromatographic assay, 100 μl (about 3 drops) of the mixture was added into a sample well for 10–15 minutes. Samples with positive control and test lines were determined as M. pneumoniae positive; no Control line on the plate indicated an invalid test, and a second test plate was used till the result was either positive or negative.

This study was supported by Key Projects of the National Science & Technology Pillar Program (2012BAI04B05) and Traditional Chinese medicine of Zhejiang province science and technology plan project (2010ZB084).