Establishment and application of a multiple cross displacement amplification combined with nanoparticles-based biosensor method for the detection of Bordetella pertussis

Background Bordetella pertussis is the causative agent of pertussis, a respiratory tract infectious disease. Efficient techniques for detection of B. pertussis isolates are important for clinical diagnosis. Multiple cross displacement amplification (MCDA), a novel isothermal amplification based molecular detection method, has been developed to overcome the technical drawback of the current methods in recent years. This aim of this study is to develop a MCDA with Nanoparticles-based Lateral Flow Biosensor (MCDA-LFB) for the detection of B. pertussis. Results A set of 10 primers based on the pertussis toxin (PT) promoter region sequence of B. pertussis was designed. The B. pertussis-MCDA-LFB assay was successfully established and optimized at 64 °C for reaction of 40 min. The detection limit was determined as 10 fg/reaction of pure DNA, and no cross-reactions to non-B. pertussis strains were observed, based on the specificity validation. The whole operation, ranging from template preparation to result reporting, could be completed within 70 min without requirement of costly equipment. The B. pertussis-MCDA-LFB in clinic sample detection yielded identical positive rates with traditional culture and showed higher sensitivity than conventional PCR. The results of MCDA-LFB are easier to read due to the usage of LFB. Conclusions The isothermal amplification based MCDA-LFB established in the present study is a specific, sensitive, rapid and economical technique for the detection of B. pertussis.


Background
Pertussis, also referred whooping cough or named as "cough of 100 days", is a respiratory tract infectious disease primarily leaded by the causative agent Bordetella pertussis [1], a gram negative bacterium which can attach to the ciliated cells and colonize the human upper respiratory tract [2]. It can damage the respiratory epithelium by producing multiple toxins and leads to systemic effects such as the lymphocytosis [3], local inflammatory changes in the mucosal lining of the respiratory tract [1]. Whooping cough can be life threatening for newborns and young children without vaccination. B. pertussis can also infect other population such as adults, and a shift of infection cases from schoolage children to adolescents, adults and infants has been observed in the last decade [2,3]. Although the efficacious pertussis vaccines has been widely used, whooping cough results in about 200,000 deaths annually and at least 24 million new pertussis cases were reported in 2014 in children under 5 years old [2]. Most deaths caused by pertussis are from developing countries, with majority of cases in children under the age of 5 months [4]. According to the estimation of WHO in 2013, B. pertussis infection caused about 60,257 deaths in children younger than 5 years [5].
The appropriate use of clinically accurate diagnostic tests is essential for the effective diagnosis of pertussis. Pertussis can be diagnosed by the commonly used culture and serological methods [6]. However, 7 to 10 days are needed to isolate and identify, B. pertussis isolates using conventional culture method, while the indirect serological diagnosis needs about 1 month based on the reaction with sera, acute-and convalescent-phase sera [7]. As a result of the shortage of culture and serologic diagnosis, the detection results are usually not available even till the patient has already recovered from the illness. In order to improve the detection duration and sensitiveness of laboratory diagnosis, PCR-based methods have been used for the detection of B. pertussis. Most available diagnostic PCR assays, including nested PCR and real-time PCR, merely developed based on fewer targets, which cannot differentiate B. pertussis from other Bordetella spp. including B. holmesii [8]. For example, real time-PCR based on part of insertion sequences (IS) cannot be used for specific species detection, because IS481 also exists in the genome of some B. bronchiseptica strains and B. holmesii strains while IS1001 is proved in some B. bronchiseptica and B. parapertussis (Martini, et al., 2017). Other targets including sequence of BP283, BP485, ptxS1 and the pertactin genes have been used for the detection of B. pertusis, but cross-reactivity with other Bordetella spp. have been reported [9]. It has been reported that the pertussis toxin (PT) promoter sequences differ from the sequence of B. pertussis, B. parapertussis and B. bronchiseptica [10,11], which enable the feasibility to develop PCR based methods targeting the PT promoter region for the specific detection of B. pertusiss. Previous study reported that PCR based on the PT promoter sequence specifically recognize B. pertussis, but the sensitivity is insufficient [12,13]. More recently, loop-mediated isothermal amplification (LAMP) targeting the PT promoter region has been established to amplify the DNA of B. pertussis with high degree of simplicity and specificity [7]. Although LAMP assays showed high efficiency of amplification samples with marginal amounts of DNA were still difficult to detect [14]. Particularly, the results of LAMP techniques for B. pertusiss detection were determined using agarose gel electrophoresis, real-time turbidity equipment or color indicator. The operation of gel electrophoresis for the analysis of products is complicated, and the risk of DNA product degradation and carryover contamination is increased [15]. The reading of real-time turbidity of B. pertussis-LAMP amplification requires optical instrument, and is easily to be interfered by the background. The judgment of result of B. pertussi-LAMP using the naked eyes is possibly subjective, which is also possible that the result reading be ambiguous to the naked eye due to the low concentration of DNA template.
In recent years, multiple cross displacement amplification (MCDA), a novel isothermal amplification based molecular detection method, has been developed to overcome the technical drawback of the current methods [16,17]. MCDA designed ten primers (two displacement primers, two core primers and six amplification primers), instead of two in PCR and six in loop-mediated isothermal amplification assay, to recognize 10 different regions of target sequence, which enhance its sensitivity, specificity, and shorten its reaction time. Moreover, MCDA assay requires merely isothermal conditions and simple equipment such as water bath or heater. The amplification products can be detected by using disposable lateral flow biosensors, with visual result judgment [18], which is objective and does not need any instrument for result reading. MCDA has been validated for the effective detection of various bacteria such as Klebsiella pneumonia, Staphylococcus aureus and Pseudomonas aeruginosa [16,17]. However, MCDA-LFB has not been validated for the detection of B. pertussis. Herein, MCDA targeting the PT promoter region, combined with nanoparticles-based biosensor, was established and optimized for simple, rapid, high specific and sensitive detection of B. pertussis at the current report, which was further evaluated by comparison with traditional culture and PCR methods.

Results
Confirming the effectiveness of B. pertussis-MCDA primer set To confirm the effectiveness of B. pertussis-MCDA primer set targeting on the PT promoter region (Fig. 1, Table 1), the DNA extracted from B. pertussis strain was amplified with MCDA at 64°C for 1 h. Both the colorimetric indicator (MG) and LFB showed that DNA of B. pertussis strain ATCC-9340 was amplified effectively, but no amplification of DNA products was observed for S. aureus (GZCDC isolate), N. meningitidis (GZCDC isolate) and DW (blank control) ( Fig. 2a and b). The electrophoresis gels images, showing a DNA ladder, were observed in positive reaction, but no DNA ladder was observed in the negative and blank controls (Fig. 2c). Therefore, the MCDA primer set was selected as the candidate to establish MCDA-LFB assay for the detection of B. pertussis.
Optimizing the reaction temperature of B. pertussis-

MCDA-LFB
To optimize amplification temperature, genomic DNA templates from B. pertussis ATCC-9340 were applied as the positive control with 10 pg DNA for each reaction. The real-time turbidity of amplification products was monitored. Typical kinetics graphs were observed for all the determined temperatures (from 60 to 67°C with increments of 1°C), and faster amplification was achieved at 64°C (Fig. 3). Therefore, 64°C was selected as the amplification temperature for the further experiments.

Sensitivity of MCDA-LFB for B. pertussis detection
Serially diluted genomic DNA from B. pertussis was used to examine the detection limit of the B. pertussis-MCDA-LFB. The results detected with LFB showed that the sensitivity of the B. pertussis-MCDA-LFB was as low as 10 fg (2.4 copies) per reaction (Fig. 4a). By MG, the detection limit of the B. pertussis-MCDA assay was also 10 fg per reaction (Fig. 4b), which was in accordance with the detection results of LFB.   Table 1 The primers used in this study Primers name a Sequences and modifications b Length c Gene

Specificity of B. pertussis-MCDA-LFB assay
The initial specificity of the MCDA primers for B. pertussis detection was confirmed by blast analysis in NCBI data base and the PT promoter sequence were only found in B. pertussis strains, with similarity of 99.5%~100%. Then, genomic DNA template from B. pertussis and non-B. pertussis strains (Table 2) were used to assess the specificity of B. pertussis-MCDA-LFB Assay. The results showed that two red lines appeared at the location of TL and CL on the strips for the B. pertussis strain, but only one line appeared at the location of CL for all the non-B. pertussis strains and blank control ( Fig. 6), suggesting negative results for non-B.
pertussis bacterial isolates and DW sample (blank control).

Application of B. pertussis-MCDA-LFB in clinical samples
In order to evaluate the B. pertussis-MCDA-LFB for clinical application, 67 nasal swab samples were used for DNA template preparation, which were were applied for the evaluation of  (Table 3).

Discussion
It has been reported that 156 million cases of pneumonia each year in children younger than 5 years are estimated by the World Health Organization (WHO) [19,20]. Recent evidence shows a sizable infant cases of pertussis present with acute pneumonia and 2% of clinical pneumonia cases of enrolled infants were caused by pertussis [20,21]. B. pertussis is the major causative reagent of pertussis, albeit other Bordetella bacteria such as B. parapertussis and B. holmesii [22] can cause less severe symptoms. Accurate and timely diagnosis of pertussis is extremely important, but the diagnosis of pertussis and accurate laboratory detection of Bordetella infections is still challenging. For instance, the commonly used culture of B. pertussis is fastidious, with limited sensitivity [23], while previously developed PCR-based methods of B. pertussis have been confirmed with the shortage of low sensitivity or cross-reaction with other Bordetella bacteria strains [8,9,12,13].
In this study, MCDA-LFB based on the PT promoter region of B. pertussis, with excellent specificity, was successfully established. The high specificity of B. pertussis-MCDA-LFB is likely due to applying PT promoter region as detection target [12,13]. Furthermore, the primer set based on different sequence of the promoter region also contributed the high degree of specificity of B. pertussis-MCDA, which has been previously demonstrated [24]. Particularly, the MCDA primer set, which recognized 10 regions of PT promoter, also ensured the assay's specificity. Although several closely related species (such as B. bronchiseptica, B. holmessii and B. hinzii) did not be examined to validate B. pertussis-MCDA- LFB's specificity, the data of specificity from NCBI BLAST has revealed that the MCDA primer set designed here was specific to B. pertussis. Furthermore, the specificity of the assay was further evaluated by using non-B. pertussis strains (Fig. 6) and clinical samples (Table 3). Therefore, the B. pertussis-MCDA-LFB established in the present study is of high degree of specificity. Except the advantage of specificity, the B. pertussis-MCDA-LFB established in the present study also displayed excellent sensitivity. The detection limit for B. pertussis pure cultures reached as low as 10 fg DNA per reaction (Fig. 4). Compared with the official real-time PCR assay recommended by China CDC, the B. pertussis-MCDA-LFB displayed better sensitivity because the limit of detection of real-time PCR kit was 70 copies per reaction (equivalent to 292 fg per reaction) provided by its manual. Furthermore, the application of detection for B. pertussis in clinical samples also verified the outstanding sensitivity of the B. pertussis-MCDA-LFB, which yielded higher positive rates than real-time PCR assay ( Table 3). The lower detection rate of real-time PCR may be due to the factors that the copy numbers of the B. pertussis templates were lower than LoD (limit of detection), or the presence of some inhibitors specific to real-time PCR decreased the reaction sensitivity.
Fast diagnosis of the pathogenic agents contributes to the accurate clinical diagnosis and rational therapy of patients. Traditional culture method needs more than 1 week to determine B. pertussis in the clinical sample, while serological diagnosis needs about 1 month [7,25]. MCDA-LFB assay developed in this study can be finished within 70 min, which includes 25 min for sample preparation, 40 min for amplification reaction and 2 min of results judgment. Lateral flow biosensor used for result interpretation in this study is time-saving, simpler compared with other methods such as traditional culture, PCR based methods or LAMP. Besides, result interpretation with LFB is based on the test line and control line, thus it is more objective and less error-prone compared with traditional culture, PCR based methods or LAMP.
Detection cost may be an economic burden for many laboratories, particularly in developing countries, which may influence the timely diagnosis for patients. The B.    pertussis, which is of potential application value in the field and resource-poor laboratories.

Reagents and instruments
The

Nanoparticle-based biosensor
Lateral flow biosensor (LFB, 4 mm × 60 mm) used in this study was prepared according to previously reported operation with some modifications [26]. Briefly, a backing card was laminated with a sample pad, absorbent pad conjugate pad and NC membrane. The test line (TL) and control line (CL) were prepared by spraying NC membrane with anti-FITC Ab (0.25 mg/ml) and biotin-BSA (2.5 mg/ml), with separation of 5 mm between the TL and CL. The conjugate pad of the strip was then coated with streptavidin which was coated with polymer nanoparticles resolved in 0.01 M PBS (PH 7.4). The prepared cards were sliced into 4-mm-wide strips (Deli No. 8012) and packed with plastic bag accompanied with desiccant gel and stored at RT (room temperature).
The genomic DNA of non-B. pertussis strains ( Table 2) were extracted with the QIAamp DNA Mini Kit (Qiagen, Germantown, MD, USA).

B. pertussis-MCDA primer set design and synthesis
The PT promoter sequence targeting B. pertussis was applied for MCDA primer design. A set of 10 primers (F1, F2, CP1, CP2, C1, C1*, C2, D1, D2, R1* and R2) was designed by using PREMIER 5.0 to establish MCDA assay. Integrated DNA Technologies design tools were applied to analyze the hairpin structures and hybrids of primer sequences. Specificity of the MCDA primers for B. pertussis detection was confirmed by blast analysis in NCBI data base. The biotin and fluorescein isothiocyanate (FITC) was used to label the 5′ ends of C1 and R1 primer, individually. The primer information is shown in Fig. 1 and Table 1. All the primers of HPLC purification grade were produced by Tianyi-Huiyuan Biotech Co., Ltd. (Beijing, China).
The B. pertussis-MCDA reaction MCDA reactions were performed in 25 μl reaction system as previously described [30]. The reaction system for each sample contained 12.5 μl of 2 × reaction mix (isothermal amplification® kit), 0.4 μM of primer F1 and F2, 0.8 μM of primer C1 * , C2, R1*, R2, D1 and D2, 1.6 μM of cross primer CP1 and CP2, 1 μl of Bst 2.0 DNA polymerase (8 U) and 1 μl of DNA template. MG and LFB detection were simultaneously applied to monitor the MCDA amplification products. By using the MG method, color changes from colorless to light green as a result of the reaction products should be detectable while no color changes observed in the negative and blank control. When using the LFB for product detection, two visible lines should be observed in positive reactions at the location of CL and TL, respectively, but only one line at the location of CL appear in the blank controls and negative. By confirming the target-specific formation of MCDA products by electrophoresis gels images, a DNA ladder should be observed in positive reaction, but no DNA ladder observed in the negative and blank control.
Optimizing the amplification temperature of B. pertussis-

MCDA-LFB
Real-time turbidity determination was used to monitor the MCDA reactions for detecting the DNA B. pertussis. The optimum reaction temperature was determined using DNA of B. pertussis from 60°C to 67°C for 60 min, with interval of 1°C. Turbidimeter (LA-320C) was used to monitor the turbidity of MCDA amplification. Genomic DNA (1 μl) of S. aureus and N. meningitidis strains were used as negative controls, and doubledistilled water (DW) were chosen as blank controls.

B. pertussis-MCDA-LFB sensitivity determination
The serial dilutions of of B. pertussis genomic templates described above were used to determine the limit detection of B. pertussis-MCDA assay. LFB and colorimetric indicator (MG) were used to detect the MCDA amplification results, respectively.

Optimizing the amplification time of B. pertussis-MCDA-LFB
The serially diluted DNA was used to optimize the MCDA reaction time. MCDA reaction mixture was incubated for 10, 20, 30 or 40 min at the optimal temperature. The MCDA amplification products were detected with a LFB, Test of each amplification time were repeated at least two times.

B. pertussis-MCDA-LFB specificity determination
The genomic DNA templates of 17 B. pertussis strains and 27 non-B. pertussis strains were applied to validate the specificity of B. pertussis-MCDA. LFB was used to detect the amplification results ( Table 2). The examinations for MCDA specificity validation were repeated at least two times.

Application of the B. pertussis-MCDA-LFB in clinical samples
A total of 67 nasal swab samples, collected from patients distributed in the prefecture Guiyang, Anshun, Zunyi, Tongren, Qiannan, Qiandongnan Qianxinan and Bijie of Guizhou Province, were used for the clinical validation of B. pertussis-MCDA-LFB. Particularly, the clinical samples used in this study, which were part of the routine CDC (Center for Disease Control and Prevention) laboratory procedure in China, were not specifically isolated for this research. The National Health and Family Planning Commission of China determined that the collection of data from human cases of infectious disease was part of continuing public health surveillance of a notifiable infectious disease and was exempt from institutional review board assessment [31]. All data were supplied and analyzed in an anonymous format, without access to personal identifying information. The total genomic DNA templates from the clinical samples stored in − 70°C for further use after regular detection by traditional culture and a real-time PCR assay (Recommended by China CDC) were used for the evaluation of B. pertussis-MCDA-LFB in clinic detection. The specificity and sensitivity of B. pertussis-MCDA-LFB, traditional culture and the conventional real-time PCR for the detection of the genomic DNA of B. pertussis from the clinical samples were compared. In particular, the B. pertussis-RT-PCR assay recommended by China CDC (MABSKY Biotech Co., Ltd., Beijing, China) was conducted according its manual ( # SKY-PCR-FZ-300-1). Briefly, each 25 μL reaction mix contained 12.5 μL of 2× Reaction Mix, 2 μL of primer mix, 5.5 μL of deionized water and 5 μL of templates. Amplification was carried out as the following conditions: 95°C for 3 mins, followed by 40 cycles of 95°C for 5 s, 55°C for 40 s.

Availability of data and materials
The datasets supporting our findings are included in the article.
Ethics approval and consent to participate These clinical samples used in this study were not specifically isolated for this research. They were part of the routine CDC (Center for Disease Control and Prevention) laboratory procedure in China. The National Health and Family Planning Commission of China determined that the collection of data from human cases of infectious disease was part of continuing public health surveillance of a notifiable infectious disease and was exempt from institutional review board assessment. All data were supplied and analyzed in an anonymous format, without access to personal identifying information. Therefore, the need for consent was deemed unnecessary.

Consent for publication
Not applicable.

Competing interests
All the authors listed in the paper declare that they have no competing interests for publication of this paper.