| BackgroundPertussis(whooping cough)is an acute respiratory infectious disease caused by Bordetella pertussis,mainly transmitted through droplets,highly infectious,susceptible to anyone of any age,and more sensitive to infants,which is an important cause of infant morbidity and mortality worldwide.The number of children cases of pertussis and deaths has declined sharply since World Health Organization(WHO)proposed Expanded Program on Immunization(EPI)in 1974.But in recent years,several developed countries with high vaccination rates have reported that the incidence of pertussis has increased again after years of low levels,such as the United States,the United Kingdom,the Netherlands,Australia and so on.Asymptomatic transmission of pertussis is one of its important causes.Asymptomatic people infected with pertussis are transmitted to susceptible people and caused their morbidity or mortality.However,the carriage rate and epidemiological characteristics of asymptomatic pertussis in the population are not clear,because the severity of clinical symptoms is positively correlated with bacterial loads,and the asymptomatic carrying bacterial load in healthy population is low,but the current laboratory detection method can't meet the requirements of low load detection and quantitative bacterial load.Droplet digital PCR(ddPCR)technology as the third generation PCR technology,without external reference standard and standard curve,can directly count or use the law of Poisson Distribution to calculate the target gene copy number to achieve really absolute quantification.Its repeatability,accuracy and sensitivity have irreplaceable advantages,especially suitable for the quantification of low load pathogens.Accordingly,the main purpose of this study was to establish an absolute quantitative method for the detection of Bordetella pertussis in healthy population based on ddPCR technologyObjectives1 To establish a new method to detect the Bordetella pertussis based on ddPCR technology and evaluate its specificity,sensitivity,repeatability and accuracy2 To compare the quantitative results of ddPCR method with traditional bacterial culture method and Real-time quantitative polymerase chain reaction(qPCR)method for Bordetella pertussis3 To explore the application of the ddPCR method for detecting of Bordetella pertussis in healthy population samples and clinical samplesMaterials and methods1 Bacterial strain and plasmidThe Bordetella pertussis strains(ATCC9797)were provided by the National Institute for Communicable Disease control of Prevention of Chinese Center for Disease Control and Prevention;The single copy IS481 reference plasmid containing Bp target genes was synthesized by Beijing Qingke Biotechnology Co.,Ltd2 SamplesNasopharyngeal swabs were collected from healthy villagers without fever,cough and other symptoms in Zhaotong City,Yunnan Province in December 2018 Oropharyngeal swabs of suspected pertussis cases were collected in Hebei Children's Hospital from August 2018 to March 20193 Methods3.1 Bacterial culture methodsNasopharyngeal swabs were collected from healthy villagers without fever,cough and other symptoms in Zhaotong City,Yunnan Province in December 2018.The selection criteria are as follows:1 No symptoms of fever,sore throat,cough in the last month2 No antibiotics and antiviral drugs used in the last month3 No other acute or chronic respiratory diseasesThe stratified random sampling method was used,to divided the samples into seven age groups(<1 year,1-2 years,3-5 years,6-17 years,18-24 years,25-54 years and?55 years),with 30 subjects in each group.Two nasopharyngeal swabs of the study subjects were collected by the same professionally trained staff.One nasopharyngeal swab of one side nostril was first rotated and smeared in the first zone of culture medium(1/3 size),and then inoculated with selective Charcoal Agar Medium(containing 10%goat blood)by three-zone line method with inoculation ring After inoculation,the culture medium was transported to the laboratory by heat preservation and placed in a pure oxygen incubator.The traditional separation culture method was carried out and identified by Gram staining,qPCR method and serum agglutination test3.2 qPCR3.2.1 Sample processing1 Swabs of health people:other nasopharyngeal swab collected from Zhaotong City were immediately transported back to the laboratory(within 4 hours).Swab was put in one tube with 600?l saline(0.85%)and repeated twisting for 50-80 times.Then we centrifuged the eluent 12,000 rpm for 5min,removed the supernatant,added 100?l of deionized water to heat in a 100? metal bath for 10 min,and centrifuged the eluent for 12,000 rpm for 5.Taking the supernatant and using fluorescence quantitative PCR to detect the CT value.Stored in-80?.2 Swabs of suspected pertussis cases:Oropharyngeal swabs collected from Hebei Children's Hospital immediately were transported back to the laboratory(within 4 hours).Swab was put in one tube with 600?l saline(0.85%)and repeated twisting for 50-80 times.Then we centrifuged the eluent 12,000 rpm for 5 min,and removed the supernatant.Total DNA was extracted using the QIAamp DNA Mini Kit Stored at-80?.3.2.2 Detecting samplesUsing qPCR to detect the CT value of swabs in 3.2.1.The CT<38 of healthy population samples were positive;the CT<35 of suspected pertussis case samples were positive.3.3 ddPCR to detect Bordetella pertussis3.3.1 Establishment of methodThe detection method of pertussis abalone based on ddPCR was established systematically.The annealing temperature,primer and probe concentration were optimized3.3.2 Evaluation methodThe specificity of the method was evaluated using the DNA of other common respiratory bacteria in China;The sensitivity and accuracy of the method was evaluated by using IS481 single copy plasmid;The repeatability of the method were evaluated by using the pure bacteria with 10 times ratio dilution DNA3.4 Comparison between ddPCR,bacterial culture and qPCR methods in detecting Bordetella pertussisPrepared of Bordetella pertussis suspension(UV600=0.5 O.D)after 10 times dilution,100?l were cultured continuously in Charcoal Agar Medium and counted colony count after five days.QlAamp DNA mini kit was used to extract the suspension of Bordetella pertussis bacteria(UV600=0.5 O.D)DNA,and the quantitative results of DNA after 10 times dilution were detected by using ddPCR and qPCR.3.5 Application of ddPCR methods in samplesNasopharyngeal swab samples in healthy population and clinically suspected pertussis samples were detected using ddPCR and qPCR method,and analyzed and compared their application in sample detection4 Data processing and analysisCT values and copy numbers were expressed using mean and standard deviation;intra-and inter-repeatability were expressed by coefficient of variation(CV).The correlation between the results of ddPCR and the culture and the correlation between the logarithmic value of the ddPCR results and the qPCR results were analyzed by linear regression analysis.The correlation coefficient R2 were calculated.P<0.05 were statistically differentMain results1 This study systematically established a ddPCR method for the detection of Bordetella pertussis.The annealing temperature was 51.1?,the primer concentration was 400 nM,and the probe concentration was 200nM.The specificity,sensitivity,repeatability and accuracy were evaluated.To evaluate the specificity of ddPCR method,we detected DNA of Streptococcus pneumoniae,Haemophilus influenzae and Streptococcus pneumoniae and water.And the results were all negative and no specific amplification.The lowest limit of detection of this ddPCR method was 1copy per reaction;The coefficient of variation between groups and in groups were 1.62%and 0.89%.The ddPCR detection range was six orders of magnitude.In the concentration range of plasmid reference 4.0 copies/?l-4.0×04 copies/?l,the theoretical copy number of the target gene was linearly correlated with the copy number detected by ddPCR and R2 was 0.99982 In the detection of nasopharyngeal swabs of healthy population using ddPCR,one copy per reaction was considered to be criterion for detecting asymptomatic pertussis.The positive swabs including 4 samples in CT<35,7 samples in 35?CT?38,7 samples in 38?CT?40,11 samples in CT?40.CT<38 was considered to be criterion for detecting asymptomatic pertussis using qPCR.The rate reached to 76.32%(29/38)using ddPCR and the rate was 28.95%(11/38)using qPCR3 In the detection of clinically suspected pertussis cases using ddPCR,the positive swabs including 40 in 40 samples in CT<35,25 in 26 samples in 35?CT<38,8 in 9samples in 38?CT<40,5 in 12 samples in CT?40.CT<35 and initial concentration of sample>29.4 copies/?l were considered to be criterion for detecting asymptomatic pertussis using qPCR and ddPCR.The rate reached to 51.72%(45/87)using ddPCR and the rate was 45.98%(40/87)using qPCR.The Kappa value of the two methods was 0.89,which had high intensity consistency.Conclusions1 The detection method of Bordetella pertussis based on QX200 ddPCR technology established in this study system has good specificity and accuracy,and is more sensitive than the traditional bacterial culture method,qPCR method2 The ddPCR method is more sensitive than the bacterial culture method,qPCR method.It is more suitable for the detection of asymptomatic pertussis,and our study provides a new method for the detection of asymptomatic pertussis3 DdPCR method is more sensitive than qPCR method in the detection of clinical sample,and can detect those infected with Bordetella pertussis in laboratory diagnosis of non-pertussis cases4 It is meaning and important to establish other common respiratory bacteria such as Streptococcus pneumoniae,Haemophilus influenzae and Streptococcus pneumoniae based on droplet digital PCR technology. |
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