Establishment and Performance Evaluation of Multiplex PCR-Dipstick DNA Chromatography for Mycoplasma pneumoniae and Chlamydia pneumoniae Rapid Detection

Methods Nasopharyngeal swab samples of 300 children with an acute respiratory tract infection were detected by a multiplex PCR-dipstick chromatography assay, and the results were compared with the DNA sequencing and serum IgM antibody assay. Results A multiplex PCR-dipstick DNA assay can specifically detect Mycoplasma pneumoniae and Chlamydia pneumoniae and shows a good specificity, with a minimum detection limit of 10 CFU/mL, respectively. Using DNA sequencing results as the gold standard, the sensitivity, specificity, positive predictive value, and negative predictive value of the multiplex PCR-dipstick DNA chromatography assay for the diagnosis of Mycoplasma pneumoniae were 96.61%, 100%, 100%, and 99.18% respectively, and those of Chlamydia pneumoniae were 95.24%, 100%, 100%, and 99.64% respectively. There was no statistical significance MP and CP diagnosis by the multiplex PCR-dipstick DNA assay and DNA sequencing (MP: P = 0.5; CP: P = 1.0), and the two assays had very high statistical consistency (MP: kappa = 0.979; CP: kappa = 0.974). The positive rate of the multiplex PCR-dipstick chromatography assay was significantly higher than that of the serum IgM antibody assay, with MP (17.7% vs. 13.3%), CP (5.7% vs. 3.3%), and mixed infection of MP and CP (1.3% vs. 0.67%). Conclusions A multiplex PCR-dipstick chromatography assay was successfully established for the joint detection of Mycoplasma pneumoniae and Chlamydia pneumoniae within 2 hours. It is simple, fast, sensitive, accurate, cost-effective with good diagnostic performance, which can be used for small laboratories and point-of-care diagnosis.


Introduction
Acute respiratory tract infections (ARTIs) are one of the main causes of hospitalization and death in children under 5 years old [1].At present, respiratory viruses are still the most common pathogens [2], but Mycoplasma pneumoniae (M.pneumoniae, MP) and Chlamydia pneumoniae (C.pneumoniae, CP) infections are becoming increasingly prominent in respiratory infections, causing approximately 10-40% and 5-10% of children's infections, respectively, and there are 1-2% mixed infections of M. pneumoniae and C. pneumoniae [3][4][5].Te clinical symptoms of infection with M. pneumoniae and C. pneumoniae lack specifcity, and empirical treatment with macrolide antibiotics may lead to an increase in the drug resistance rate.Currently, the drug resistance rate of M. pneumoniae ranges from 85% to 100% [6][7][8].Terefore, it is important to have a rapid and accurate laboratory diagnosis before conducting antibacterial treatment.Te widely used identifcation methods in clinical include isolating culture, serum antibody testing, and molecular biology.M. pneumoniae rapidly proliferates in the culture medium, decomposes glucose to produce acid, and changes the pH value of the culture medium so that M. pneumoniae can determine the culture positive by observing the color change of the culture medium.However, M. pneumoniae cultivation conditions are harsh, timeconsuming, and the positive rate is low [9,10], which cannot meet the needs of rapid clinical diagnosis.C. pneumoniae is a microorganism that grows within cells and cannot be cultured in vitro.For the serum antibody detection method, MP-IgM and CP-IgM increase around 1 week of onset, reaching a peak at 3-6 weeks [11].In the acute phase (within 1 week of onset), MP-IgM is found to be positive in 14%-45% patients [12], so this method cannot achieve the detection purpose in the early stage of infection.Te exact sensitivity and specifcity of PCR methods vary with the corresponding amplifcation platform and detection form [13]. Routine PCR needs to be analyzed in agarose gel electrophoresis, which takes a long time and has low sensitivity.Te real-time fuorescent PCR method has high sensitivity and good detection specifcity, but the requirements for complex equipment and well-trained personnel make it difcult to be widely used in small community hospitals and point-of-care diagnosis [14].Loop-mediated isothermal amplifcation (LAMP) technology is a relatively new form of nucleic acid amplifcation testing, widely used for direct detection of clinically important pathogens, including M. pneumoniae [15].However, traditionally, detecting turbidity changes in reaction mixtures requires the use of a spectrophotometer, which will limit its widespread use in restricted environments.Although visual detection reagents (VDRs) are an alternative method, the results may become less accurate at low template concentrations [16].PCR-based DNA biosensors have been designed for the detection of pathogens and drugresistant genes in the form of test strip nucleic acid detection, with advantages such as rapidity, specifcity, sensitivity, low detection limit, and cost-efectiveness [17][18][19][20][21]. Tis study aimed to establish a multiplex PCR-dipstick DNA chromatography assay for the joint detection of M. pneumoniae and C. pneumoniae based on PCR and strip DNA biosensors.

Sample Preparation.
Te nasopharyngeal swab sample was placed in 1 mL sterile physiological saline, stirred 20 times, and centrifuged at a speed of 15,000 rpm for 5 minutes, and then, the supernatant was discarded as much as possible.According to the manufacturer's instructions, the Magen HiPure bacterial DNA kit was used to extract genomic DNA content from nasopharyngeal swabs.Te serum samples were centrifuged at a speed of 3,000 rpm for 5 minutes and sent to the laboratory for MP-IgM and CP-IgM testing.
2.4.Primer and Probe Design.Primers were designed according to the conserved sequence of MP and CP using prime 5.0.Te 5′ end of the forward primer is labeled with oligonucleotide (Tag), the 5′ end of the reverse primer is labeled with biotin, and the forward primer end is connected to Tag with spacer C3, which can ensure that Tag remains single-stranded during amplifcation.In order to verify the DNA extraction efciency and reaction performance of the sample, an internal control (IC) was designed, which is a synthetic sequence with extremely low sequence homology and will not cross react with other genomes.A pair of primers was also designed for IC.Te primers and the probes (Table 1) were synthesized by TBA (Tohoku Bio-Array, TBA, Sendai, Japan).

Sensitivity of Multiplex PCR-Dipstick Chromatography.
In order to assess the limit of detection (LOD) of multiplex PCR-dipstick chromatography, serial 10-fold dilutions of M. pneumoniae and C. pneumoniae (10-10 7 CFU/mL) were analyzed by multiplex PCR-dipstick DNA chromatography to determine the sensitivity.M. pneumoniae and C. pneumoniae sensitivity of the PCR-dipstick chromatography assay was 10 CFU/mL, respectively (Figure 3).

Data of Clinical Specimens.
Nasopharyngeal swab samples of 300 children with an acute respiratory tract infection were detected by the multiplex PCR-dipstick chromatography assay and DNA sequencing.Meanwhile, blood samples from each child were identifed by the MP-IgM and CP-IgM antibody assay.As shown in Tables 2 and  3, among the samples from children with an acute respiratory tract infection, 53 (17.7%) of them were positive for MP, 16 (5.3%) of them were positive for CP, and 4 (1.3%) of them were simultaneously positive for MP and CP by the multiplex PCR-dipstick chromatography assay.DNA sequencing showed that 55 cases (18.3%) were MP positive, 17 cases (5.7%) were CP positive, and 4 cases (1.3%) were simultaneously positive for MP and CP.Tere was no statistical signifcance for MP and CP detection between the multiplex PCR-dipstick chromatography assay and DNA sequencing (MP: P � 0.5; CP: P � 1.0), and a high statistical consistency was observed between the two assays (MP: kappa value � 0.979; CP: kappa value � 0.974).
Blood samples of all children were tested by the MP-IgM and CP-IgM assay.Among 300 samples, 40 cases (13.3%) were positive for MP-IgM, 10 cases (3.3%) were positive for CP-IgM, and 2 cases (0.67%) were simultaneously positive for MP-IgM and CP-IgM.Te multiplex PCR-dipstick chromatography assay for detecting MP and CP showed statistical signifcance compared to serum MP-IgM and CP-IgM, with P � 0.001 (MP) and P � 0.021 (CP), respectively.Te two assays for detecting MP have a high degree of agreement, with a kappa value of 0.747.Te agreement for detecting CP is average, with a kappa value of 0.671.

Discussion
Atypical pneumonia caused by M. pneumoniae and C. pneumoniae has similar respiratory manifestations to bacterial and viral infections, which is prone to misdiagnosis and missed diagnosis.Terefore, rapid, sensitive, and specifc detection methods are necessary for clinical diagnosis.In order to improve laboratory diagnosis and reduce the cost and time of detecting M. pneumoniae and C. pneumoniae, we designed and validated a multiplex PCR-dipstick chromatography assay that can simultaneously detect M. pneumoniae and C. pneumoniae.Te results indicated that we successfully developed a multiplex PCR-dipstick chromatography assay in this study to achieve rapid detection of M. pneumoniae and C. pneumoniae within 2 hours.Real-time fuorescence quantitative PCR detection is often used to identify M. pneumoniae due to its high sensitivity and specifcity [22,23].Te limit of detection (LOD) of multiplex PCR-dipstick chromatography for M. pneumoniae and C. pneumoniae was 10 CFU/mL, which is not lower than that of real-time fuorescence quantitative PCR.In addition, this assay has strong specifcity and does not require complex and expensive equipment.It only requires a regular PCR amplifcation instrument, which has broad prospects for promotion and application.Using DNA sequencing as the diagnostic gold standard, 300 clinical samples were analyzed by multiplex PCR-dipstick chromatography.Te sensitivity, specifcity, positive predictive value, and negative predictive value of the multiplex PCRdipstick chromatography assay for the diagnosis of M. pneumoniae were 96.61%, 100%, 100%, and 99.18%, respectively, and the sensitivity, specifcity, positive predictive value, and negative predictive value for the diagnosis of C. pneumoniae were 95.24%, 100%, 100%, and 99.64%, respectively.Te positive rate of the multiplex PCR-dipstick chromatography assay was signifcantly higher than that of the serum IgM antibody assay, with M. pneumoniae (17.7% vs. 13.3%),C. pneumoniae (5.7% vs. 3.3%), and mixed infection of M. pneumoniae and C. pneumoniae (1.3% vs. 0.67%).For 57 positive cases of M. pneumoniae detected by the multiplex PCR-dipstick chromatography assay, only 39 cases were MP-IgM positive and 18 cases were MP-IgM negative.Among the 20 positive cases of CP detected by the multiplex PCR-dipstick chromatography assay, 11 cases were CP-IgM positive and 9 cases were CP-IgM negative.Reviewing the case data of 18 patients with negative MP-IgM and 9 patients with negative CP-IgM, 12 cases of the 18 patients with negative MP-IgM had a disease course of less than one week and 6 cases had a disease course of 7-10 days.Among the 9 patients with negative CP-IgM, 6 cases had a course of less than one week, 2 had a course of 8-9 days, and 1 case had a course of 12 days.Due to the increase of IgM in about one week of onset and reaches its peak in 3-6 weeks [11], the inconsistency between the multiplex PCR-dipstick chromatography assay and serum IgM antibody levels may stem from the presence of an antibody production window during serum antibody testing.Tis window refers to a specifc timeframe in which the body either does not produce antibodies or produces them in such low concentrations that they may not be detected.In this study, three cases of MP IgM positive and one case of CP IgM positive were identifed, however, all four samples showed negative results by multiplex PCR-dipstick chromatography assay.To further investigate this discrepancy, DNA sequencing was performed on the four samples.Only one sample that had tested positive for MP IgM yielded a positive result through DNA sequencing.Tis fnding suggested that the inconsistency observed in this case might be attributed to the low concentration of the bacterium in the nasopharyngeal swab samples, which was below the detection limit of the multiplex PCR-dipstick chromatography assay.chromatography assay is that it can only be used for early infection diagnosis but not suitable for determining the clearance rate of pathogens after medication.A positive result cannot distinguish between carriers and recent or past infection status.Second, macrolide-resistant strains of M. pneumoniae and C. pneumoniae have emerged in the past few years, with a high incidence rate in China and Japan, which may lead to treatment failure.Terefore, the detection of resistance to macrolide drugs is also crucial, but this assay cannot determine drug resistance.
With the development of the signal processing, probe, and multiplex PCR technology of the strip biosensor, resistance genes to macrolide antibiotics should be incorporated in a PCR-dipstick chromatography assay.

Conclusion
Tis study successfully established a multiplex PCR-dipstick chromatography assay for the joint detection of M. pneumoniae and C. pneumoniae.It is fast, sensitive, and specifc and is expected to become a new technical means for clinical diagnosis and rapid screening, which is suitable for small laboratories and point-of-care diagnosis.
PCR-dipstick chromatography detection results were compared with MP-IgM, CP-IgM, and DNA sequencing, respectively.Te diagnostic sensitivity, specifcity, positive predictive value, and negative predictive value of the PCRdipstick chromatography assay were evaluated with DNA sequencing as the gold standard.We used the SPSS pairing χ 2 test (McNemar test) to compare the diagnostic efciency of the PCR-dipstick chromatography assay with MP-IgM, CP-IgM, and DNA sequencing, respectively.P < 0.05 indicates a statistically signifcant diference.Kappa ≥ 0.7 indicates strong ft; 0.7 > kappa ≥ 0.4 indicates average ft; kappa < 0.4 indicates weak ft.

Figure 3 :
Figure 3: Sensitivity of the multiplex PCR-dipstick chromatography assay for detection of M. pneumoniae and C. pneumoniae.(a) M. pneumoniae; (b) C. pneumoniae.1: negative control; 2-8: detection of each bacterial standard strain DNA with a concentration ranging from 10 CFU/mL to 10 7 CFU/mL, respectively.
Te PCR amplifcation reaction procedure was performed at 95 °C for 3 minutes, and the amplifcation programme for a total of 40 cycles is as follows: 95 °C for 5 s; 60 °C for 20 s.Canadian Journal of Infectious Diseases and Medical Microbiology red line is used as a marker line to locate diferent bacterial test lines.Biotin at the 5′end of the reverse primer combined with the blue latex microspheres coated with streptavidin to generate signals.Target DNA amplicons with Tag and a biotinylated terminus were coupled with latex beads through a streptavidin-biotin interaction, and then, Tag hybridized with cTag on the strip.Te accumulation of captured latex beads on the test line and IC line produced blue bands, enabling visual detection with the naked eye.After PCR PCR-Dipstick DNA Chromatography 2.5.1.Multiplex PCR Reaction.Te reaction system includes 2.5 μL 10 × PCR bufer, 5 μL DNA template, 0.5 μL forward and reverse primers of MP and CP (10 μM), respectively, 0.5 μL IC forward and IC reverse primers (2 μM), respectively, 1 μL IC DNA (1 nM), 1.2 μL RT enzyme mixture, 1 μL dUTP + dNTP mixture, 0.02 μL uracil N-glycosylase (UNG), 0.3 μL Taq HS polymerase, and 10.98 μL deionized water, in a total volume of 25 μL.2.5.2.Dipstick DNA Chromatography.Te dipstick strip, manufactured by Tohoku Bio-Array, Co., contains three primary components: a polyvinyl chloride (PVC) membrane provides stability and adhesiveness, an absorbent area made of absorbent flter paper, and a sample application area made of a nitrocellulose (NC) membrane.Complementary oligonucleotide probes (cTag) were immobilized on an NC membrane to form M. pneumoniae and C. pneumoniae test lines and an internal control line.In addition, the sample application area was printed with four red position lines.Te 2 amplifcation, 10 μL PCR products, 9 μL eluent (containing detergent, blocking agent, PBS, and salt solution), and 1 μL of streptavidin-coated blue latex were added to the test tube to form a mixture, and then, the test strip was inserted into the mixture.After 10 minutes, the corresponding test lines and IC lines turn blue, indicating that the target bacteria are positive.Ifonly the IC line shows color, it is negative.If the IC line does not show color, it indicates that the test strip is invalid.Te schematic diagram of multiplex PCR-dipstick DNA chromatography is shown in Figure1.2.6.Specifcity of Multiplex PCR-Dipstick Chromatography.Te PCR-dipstick chromatography assay was utilized to detect a concentration of 105CFU/mL of various bacterial pathogens, including M. pneumoniae, C. pneumoniae, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Haemophilus infuenzae, Streptococcus pneumoniae, Staphylococcus aureus, Acinetobacter baumannii, Acinetobacter johnsonii, Enterobacteriaceae cloacae, Klebsiella aerogenes, Haemophilus parainfuenzae, Pseudomonas fuorescens, Aeromonas hydrophila, Staphylococcus epidermidis, and Chlamydia psittaci to assess the specifcity of this assay.2.8.Clinical Sample Validation.300 nasopharyngeal swab samples were detected by the PCR-dipstick chromatography assay, and the PCR products were sent for sequencing.Te

Table 1 :
Te primers for the PCR-dipstick DNA chromatography assay.

Table 2 :
Comparison of clinical specimens of MP data between multiplex PCR-dipstick chromatography assay, DNA sequencing, and MP-IgM.

Table 3 :
Comparison of clinical specimens of CP data between multiplex PCR-dipstick chromatography assay, DNA sequencing, and CP-IgM.