Development of a Triplex qPCR Assay Based on the TaqMan Probe for the Detection of Haemophilus parasuis, Streptococcus suis Serotype 2 and Pasteurella multocida
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Porcine respiratory disease is a significant economic problem for the global swine industry.Keywords:
Streptococcus suis
TaqMan
Objective To establish a rapid detection assay of Streptococcus suis serotype 2 with Taqman-MGB probe based on real-time PCR technology.Methods By using Beacon Designer 7.0,primers and Taqman-MGB probe were designed and the real-time PCR detection assay was established to detect the csp2J gene sequence of Streptococcus suis serotype 2.The target fragment was cloned into the pMD18-T vector to make standard curve.The specificity of the probe was verified with common and conditional pathogenic bacteria,blood simulating samples were prepared to evaluate the clinical application of this assay.Results The standard curve showed that the 100 copies csp2J gene per reaction could be detected by this assay,and specificity detection revealed that only the reaction of Streptococcus suis type 2 showed the fluorescence signal,while that of other common and conditional pathogenic bacteria showed no fluorescence signal,and the lowest testing limitation of blood simulating samples was 3.9×102CFU per ml.Conclusion The real-time PCR detection assay of Streptococcus suis serotype 2 with csp2J as target gene is sensitive,specific and rapid,which can be used in preliminary screening of samples of patients infected with Streptococcus suis type 2.
Streptococcus suis
TaqMan
Standard curve
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Haemophilus parasuis and Streptococcus suis were important pathogenes of swine both causing meningitis,arthritis,endocarditis,and it was difficult to distinguish them from the clinical symptom.In this study,the sequence of Haemophilus parasuis and Streptococcus suis 16S rRNA gene were amplified by polymerase chain reaction(PCR),and 822 bp and 294 bp specific DNA fragment could be amplified.Dulex PCR with the specific primer for differentiating Haemophilus parasuis and Streptococcus suis in pigs was established,and it was conformed to be specificity and sensitivity by clinical application.This method was applied in 161 diseased pig herds Haemophilus parasuis was 23.75% positive,and Streptococcus suis was 43.48% positive.
Streptococcus suis
Haemophilus parainfluenzae
Primer (cosmetics)
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Pasteurella multocida and Haemophilus parasuis are the main pathogens of porcine respiratory disease syndrome, and often are the common pathogens of porcine pulmonary disease in collectivized pig farms. It has brought great influence on the pig industry and seriously hampered the healthy development of pigs. Therefore, the rapid detection method for Pasteurella multocida and Haemophilus parasuis is the key to its successful prevention and control. In the process of diagnosis, we need to conduct laboratory tests for confirming of preliminary diagnosis, that can be made based on the history, clinical symptoms, pathological changes of necropsy and epidemiological characteristics of the disease. Traditional diagnostic techniques, such as bacterial isolation and immunological test, are time-consuming and laborious, and are not suitable for rapid clinical diagnosis, nor for large-scale epidemiological investigation. Therefore, it is necessary to establish an accurate and rapid method to identify the two common respiratory pathogens in pigs.
In this paper, Pasteurella multocida and Haemophilus parasuis were used as research objects to establish a dual PCR detection method. The main research contents are as follows: A dual PCR assay was established to detect Pasteurella multocida and Haemophilus parasuis simultaneously. By using Pasteurella multocida and Haemophilus parasuis specific primers, and adjusting primer concentration and annealing temperature, a dual PCR method for detecting Pasteurella multocida and Haemophilus parasuis was established.
The rapid detection method for Pasteurella multocida and Haemophilus parasuis established in this study has high specificity and sensitivity, and can realize rapid and accurate identification of pathogenic bacteria in a relatively short period of time, providing a new technical means for rapid detection in clinical and grassroots laboratories
Pasteurella
Isolation
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Two real-time PCR assays for the simultaneous detection of Streptococcus suis and S.suis type 2 were developed using selective primers and TaqMan hydrolysis type probes labeled with 6-carboxy-fluorescein(6-FAM)reporter dyes.The assays were examined for sensitivity,specificity,reproducibility for detection of mocked tissue and clinical samples.The results showed that both assays were rapid,specific and sensitive.The assays could be finished in 1.5 hours(including the pretreatment of sam- ples),and the detection threshold was 10 CFU-100 CFU when used to detect the cultures and mocked tissues.
Streptococcus suis
TaqMan
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Mice and rats, free from Pasteurellaceae, were exposed to Haemophilus spp. (V-factor dependent Pasteurellaceae) by housing in proximity to infected rats or guinea pigs, and monitored by culture and enzyme-linked immunosorbent assay (ELISA) for cross infection. A minority of mice became infected when exposed to Haemophilus-infected rats but none when exposed to guinea pigs. Rats were readily infected when exposed to Haemophilus-infected guinea pigs or rats. Although Pasteurellaceae infections are commonly considered as host specific, our data show that Haemophilus spp. can cross the species barrier from rats to mice and from guinea pigs to rats.
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Multiplex-PCR detection method for Pasteurella multocida(PM) and Haemophilus parasuis(HPS) had been developed based on published single PCR for these pathogens.The PCR reagents,annealing temperature and the PCR procedure were optimized and the primers were screened,the PCR products including 457 bp of PM and 1 090 bp of HPS were obtained just by one test.The detectable minimum dose of HPS was 1.04×10~3 CFU or 7.2 pg DNA and PM was 2.3×10~3 CFU or 16 pg DNA.Among 23 suspected isolates,two strains showed PM fragments by multiplex-PCR method, no HPS fragments,but all artificially infected isolates showed HPS fragments.The results suggested that the multiplex-PCR method could be used in clinical diagnosis of PM and HPS.
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Pasteurellaceae bacteria are etiological agents of various animal diseases. Representatives of this family can be part of the "Bovine respiratory disease complex"" - a set of viral and bacterial pathogens that cause respiratory diseases in cattle. The aim of our work was to study the prevalence of Pasteurella multocida, Mannheimia haemolytica, Bibersteinia trehalosi, and Histophillus somni in calves aged 2 to 6 months in 37 farms in Moscow and Tver regions. Respiratory swabs were examined by Real time PCR. P. multocida DNA was found in all 37 farms (89% of samples). The genetic material of M. haemolytica and H. somni was detected less often - in 28% and in 18.5% of the samples. B. trehalosi DNA was detected in 3% of the samples from 8 farms. In one sample, P. multocida type F was detected, in 90.72% of positive P. multocida samples the presence of type A was confirmed. There was no circulation of P. multocida serotypes B, D, and E. Monoinfection with P. multocida was recorded only in 6 farms out of 37. In all other farms, a combination of two or more studied microorganisms was detected. An association of four pathogens was registered in four farms. The analysis of the frequency of P. multocida, M. haemolytica, B. trehalosi, and H. somni DNA detection in calves showed no correlation with the age of the animals. This may indicate that immune status and conditions of maintenance and care in the farm are of greatest importance in Pasteurellaceae prevalence in cattle.
Pasteurellosis
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Haemophilus somnus, Pasteurella haemolytica, Pasteurella multocida, and Actinobacillus pleuropneumoniae from cattle and pigs with respiratory disease were used to evaluate the RapID NH system (Innovative Diagnostics, Atlanta, Ga.). Minor modifications of the RapID NH system to include animal source and growth requirements would permit the identification of all isolates tested.
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Pasteurella
Actinobacillus
Pasteurellosis
Pleuropneumonia
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Based on partial sequencing of the 16S rRNA gene V-factor dependent Pasteurellaceae ( Haemophilus ), strains from rat and guinea pig were assigned to the Rodent cluster or the Haemophilus parainfluenzae complex. PCRs for the detection of biotype Heyl or Jawetz [ P. ] pneumotropica detected none of the strains and only two Haemophilus strains assigned to the Rodent cluster respectively. All Haemophilus strains were positive by a PCR developed for detection of all Pasteurellaceae taxa. The Pasteurellaceae PCR detected infection in all 76 rats and 40 guinea pigs from 3 and 6 colonies respectively reported to be free from Pasteurellaceae infection. ELISAs, using two Haemophilus antigens and culture, detected infection with similar frequency but both methods were inferior to PCR. The Pasteurellaceae PCR should be the new ‘gold standard’ for comparison of the sensitivity of other test methods for Pasteurellaceae infection in rodents. Â
Haemophilus parainfluenzae
Pasteurella
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A study was performed to obtain a better understanding of the diversity and ecology of members of the family Pasteurellaceae in the porcine respiratory tract. A collection of 132 V factor-dependent strains of Pasteurellaceae selected from porcine field isolates mainly from the respiratory tract were subjected to detailed characterization. In addition to the three hitherto recognized species Actinobacillus pleuropneumoniae, Haemophilus parasuis, and Haemophilus taxon "minor group," three distinct taxa were observed. Some of these taxa, which are provisionally designated taxa D, E, and F, would by traditional criteria be mistaken for H. parasuis but differed by several biochemical characteristics. To study the ecology of the V factor-dependent species, swabs from the nasal and oral cavities of 29 pigs were cultivated on selective and nonselective media. By studying approximately 30 isolates from each sample, the distribution and relative proportion of the individual taxa were determined. A. pleuropneumoniae was detected in samples from the tonsil areas of only two acutely ill animals. H. parasuis was isolated from the nasal cavities of four out of nine healthy pigs but from the oral cavities of only two animals. In contrast, taxon "minor group" and taxa D, E, and F were present in the oral cavities of the majority of pigs but were not detected in samples from their nasal cavities. The results indicate that all the observed V factor-dependent species of Pasteurellaceae except A. pleuropneumoniae, are members of the resident microflora of various mucosal surfaces of the porcine upper respiratory tract.
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Respiratory tract
Actinobacillus pleuropneumoniae
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