Development of a reliable method to diagnose Streptococcus agalactiae infection by droplet digital PCR

Background: Streptococcus agalactiae (GBS) is the causative pathogen of puerperal sepsis in pregnant women and meningitis in infants. Infection of GBS is responsible for the increased morbidity in pregnant women and the elderly, and bring challenges to clinical diagnosis and treatment. However, culture-based approaches to detect S.agalactiae is time-consuming with limited sensitivity. Besides, real-time quantitative PCR demand for expensive instruments with tedious steps. Thus, we aim to establish a new detection method for more accurate and rapid detection of S.agalactiae. Results: The ddPCR primer targeted the CspE gene showed a better amplified efficiency in the reaction. The limitation of ddPCR for identifying GBS DNA was able to reach 5 pg/µL. Moreover, no positive amplified signals could be detected in the reactions which served 11 non-GBS strains DNA as templates. Furthermore, the coefficient of variation of this method is 4.5%, indicating an excellent repeatability of ddPCR assay. Conclusions : In our study, ddPCR was performed as a rapid detection of S.agalactiae with high sensitivity and specificity. This technique can promote the accuracy of the diagnosis of GBS infection and provide a scientific basis for clinical treatment.

4 microdroplet is amplified as an independent reaction system with or without target genes. The amplified condition of ddPCR is similar to that of real-time PCR with the probe for signal detection.
Droplet digital PCR is an ultraprecise, reliable and economical method in the diagnosis of infectious disease. However, detection of GBS based on ddPCR has not been reported yet. Thus, we aim to evaluate the ddPCR for the detection of GBS and test whether ddPCR can be an alternative assay for the rapid diagnosis of GBS infection.

Primer screening test
Two sets of primes showed different amplification to GBS ATCC13813 monitored by the SLAN-96P real-time system (Figure 1). The amplified signal was firstly detected 15 cycles after the reaction and the peak emerged at approximately 45 cycles with CspE primer. No other amplification was seen in the Sip primer and negative control. Therefore, the CspE primer was selected for the subsequent tests.

Sensitivity and specificity of ddPCR for GBS
The limit of ddPCR for detecting GBS DNA was able to reach 5 pg/µL. As shown in Figure 2, the horizontal axis represented the event number of four concentrations templates, and the vertical represented the sample amplitude. The positive and negative microdroplets were shown in blue and gray, respectively. The number of events was 0 in the concentration of 0.5 pg/µL, suggesting no amplification in this reaction. (Figure 3). No positive microdroplets could be detected in the reactions using non-GBS strains DNA as templates(data not shown). Therefore, the ddPCR with CspE primer has a satisfactory sensitivity and specificity for GBS detection.

Repeatability test of ddPCR
GBS reference strain was run in triplicate ( Figure 4). The positive events number was 1661, 1560 and 1704, respectively, with a CV of 4.5%, indicating that ddPCR has an excellent repeatability.

Discussion
Streptococcus agalactiae is the leading cause of neonatal pneumonia, infantile septicemia, bacterial meningitis, as well as perinatal infection of pregnant women and infants [15,16] . Therefore, a novel detection based on ddPCR was developed to detect GBS by targeting the CpsE gene in the current study.
Nowadays, a bacterial culture is still a gold standard method to identify GBS infection, but it is timeconsuming with limited sensitivity and vulnerable to interference [17] . Also, real-time qPCR requires expensive equipment and only quantifies nucleic acid relatively. Recently, a novel technique, ddPCR, was for DNA quantifications absolutely without depending on the standard curve [18] . ddPCR is the third generation PCR with higher diagnostic efficiency compared to conventional methods. In our study, we showed that ddPCR could detect S.agalactiae precisely as low as 5 pg/µL. Moreover, amplification was observed in GBS but not in non-GBS strains, which indicated the high specificity of ddPCR primers. Furthermore, ddPCR had an excellent repeatability with a CV of 4.5%.
Given these advantages, it can be used to determine the expression and copy number variation analysis of the target gene [19,20] .
In the present study, the fluorescent dye EvaGreen was used to monitor GBS. However, EvaGreen binds to dsDNA and could cause false-positive results if there existed dimer formations. Thus, the melting curve was analyzed and no double-peak was found, indicating no primer dimer formation.
Furthermore, we discovered that the CspE primer amplifies the target gene of GBS more effectively than the Sip gene.
Some limitations of our study should not be ignored. Firstly, ddPCR is an assay mainly with fluorescent probes. Application with EvaGreen dye may significantly interfere with the experimental results when the primer dimers were forming. Therefore, the demand for primers specificity is very high. Secondly, bubbles in the process will produce less than 12 000 microdroplets. Thus the experiment does not meet the Poisson distribution, leading to inaccurate results. Thirdly, it was reported that the fbs-B gene was targeted with LAMP to identify GBS [5] . Which gene is more effective in detection needs to be further studied. Moreover, we just established a ddPCR method to identify response by droplet digital PCR. Scientific reports9, 12507, doi:10.1038/s41598-019-48966-9 (2019). Tables   Table1 Sequences of primers of Sip and CpsE gene   Target Sequences (5'-3')  Figure 1 The primer of the qPCR screening experiment in the present study.

Figure 2
Sensitivity assay for ddPCR using continuous 10-fold dilutions of DNA templates from GBS ATCC13813.

Figure 3
The number of events in each amplification concentration.

Figure 4
The repeatability test of ddPCR using DNA from the GBS ATCC13813 strain.