Development and validation of duplex and multiplex PCR assays for sensitive and rapid simultaneous detection of phytoplasma, ‘Candidatus liberibacter asiaticus’, and citrus tristeza virus associated with citrus decline disease in India

Focus Issue Articles on Diagnostic Assay Development and Validation: The Science of Getting It Right

Background: Mycoplasmosis caused by Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS), is an economically significant disease in both commercial and backyard poultry. The present work aimed at developing a duplex PCR assay for quick, accurate detection and differentiation of MG and MS and validating it for screening purpose. Methods: Duplex PCR assay was standardized with primers designed specific to the mgc2 gene for MG and hemagglutinin vlhA gene for MS with amplicon size of 150 bp and 787 bp, respectively. Validation of molecular detection of MG and MS with duplex PCR assay was performed with a total of 179 pooled oropharyngeal swab samples from backyard poultry flocks from different parts of India. Result: The duplex PCR assay showed, 133/179 (74.3%), 39/179 (21.78%) and 35/179 (19.55%) positivity for MG, MS and both MG and MS, respectively. Duplex PCR assay showed sensitivity of detection limit of 10-6 of 1 mg/ml of template DNA. Comparison of results of duplex PCR assay with single PCR of field samples showed perfect agreement between two assays (k= 0.90). The duplex PCR validated in the study would be useful in rapid screening/ surveillance, diagnosis and differentiation of MG and MS in field samples in cost-effective manner and to devise effective control strategies.

A Novel Semiquantitative Fluorescence-Based Multiplex Polymerase Chain Reaction Assay for Rapid Simultaneous Detection of Bacterial and Parasitic Pathogens from Blood

BackgroundWith the safety of blood transfusion being a major public health concern, the development of a rapid, sensitive, specific, and cost-effective multiplex PCR assay for simultaneous detection of hepatitis B virus(HBV), hepatitis C virus (HCV), hepatitis E virus (HEV), and Treponema pallidum(T. pallidum) in blood is crucial.MethodsFive primer pairs and probes were designed towards conserved regions of target genes and used to establish a one-step pentaplex real-time reverse transcription PCR(qRT-PCR) assay for simultaneous detection of HBV, HCV, HEV, T. pallidum, and RNase P(housekeeping gene), providing sample quality check. The clinical performance of the assay was further determined with 2400 blood samples from blood donors and patients in Zhejiang province, and compared the results with commercial singleplex qPCR and serological assays.ResultsThe 95% limit of detection(LOD) of HBV, HCV, HEV, and T. pallidum were 7.11 copies/µL, 7.65 copies/µL, 8.45 copies/µL, and 9.06 copies/µL, respectively. Moreover, the assay has good specificity and precision. Compared to the singleplex qPCR assay, the novel assay for detecting HBV, HCV, HEV, and T. pallidum presented 100% clinical sensitivity, specificity, and consistency. Several discrepant results between serological and pentaplex qRT-PCR assays were found. Of 2400 blood samples, there were 2(0.08%) HBsAg positive samples, 3(0.13%) anti-HCV positive samples, 29(1.21%) IgM anti-HEV positive samples and 6(0.25%) anti-T. pallidum positive samples proven negative in nucleic acid detection. 1(0.04%) HBV DNA positive sample and 1(0.04%) HEV RNA positive sample were detected negative by serological testing.ConclusionsThe developed pentaplex qRT-PCR is the first assay on simultaneous, sensitive, specific, and reproducible detection of HBV, HCV, HEV, T. pallidum, and RNase P in a single tube. It could detect pathogens in blood during the window period of infection and is a good tool for effectively screening blood donors and early clinical diagnosis.

TB or NTM: Can a new multiplex PCR assay be the answer?

Bovine babesiosis and theileriosis are fatal tick borne haemoparasites of vertebrates imposing serious constraints on health and productivity of livestock. Additionally, the recovered animals become persistent carriers and play a significant role in disease epidemiology. The present investigation describes the development and evaluation of duplex PCR assay for simultaneous detection of Babesia bigemina (B. bigemina) and Theileria annulata (T. annulata) in cattle. Following in silico analysis for candidate target genes representing each of the haemoparasites, an optimised duplex PCR assay was established using two sets of primers, ssurRNA and cytob1 for genomic DNA amplification of B. bigemina and T. annulata encoding product size of 689 and 312 bp, respectively. The results were compared with conventional microscopy and monoplex PCR assay. The sensitivity of each primer pair was checked using serial dilutions of parasite DNA, while specificity was determined by testing for amplification from DNA of different stocks of each pathogen. The duplex PCR detected each parasite species with the same level of sensitivity, irrespective of whether its DNA was amplified in isolation or with DNA mixture representing the other pathogens. Additionally, single and duplex PCRs could able to detect each species with equal sensitivity in serially diluted DNA representing mixtures of both the pathogen, and nonspecific amplification from non target species was not observed. The developed assay represents an economical, simple, sensitive, specific and reproducible diagnostic tool for simultaneous detection of tropical theileriosis and bovine babesiosis and boosting targeted selective control strategy in endemic areas.

Validation of high-throughput real time polymerase chain reaction assays for simultaneous detection of invasive citrus pathogens

Huanglongbing (HLB) or citrus greening is highly destructive disease that is affecting the citrus industry worldwide and it has killed millions of citrus plants globally. HLB is caused by the phloem limited, Gram negative, non-culturable, alpha-proteobacterium, ‘Candidatus Liberibacter asiaticus’. Currently, polymerase chain reaction (PCR) and real time PCR have been the gold standard techniques used for detection of ‘Ca. L. asiaticus’. These diagnostic methods are expensive, require well equipped laboratories, not user-friendly and not suitable for on-site detection of the pathogen. In this study, a sensitive, reliable, quick and low cost recombinase polymerase based isothermal amplification combined with lateral flow assay (HLB-RPA-LFA) technique has been developed as a diagnostic tool for detection of ‘Ca. L. asiaticus’. The assay was standardized by designing the specific primer pair and probe based on the conserved 16S rRNA gene of ‘Ca. L. asiaticus’. The assay was optimized for temperature and reaction time by using purified DNA and crude plant extracts and the best HLB-RPA-LFA was achieved at the isothermal temperature of 38°C for 20 to 30 min. The efficacy and sensitivity of the assay was carried out by using field grown, HLB-infected, HLB-doubtful and healthy citrus cultivars including mandarin, sweet orange cv. mosambi, and acid lime. The HLB-RPA-LFA did not show cross-reactivity with other citrus pathogens and is simple, cost-effective, rapid, user-friendly and sensitive. Thus, the HLB-RPA-LFA method has great potential to provide an improved diagnostic tool for detection of ‘Ca. L. asiaticus’ for the farmers, nurserymen, disease surveyors, mobile plant pathology laboratories, bud-wood certification and quarantine programs.

For many years, the pathogenic bacterium Neisseria gonorrhoeae, the etiologic agent of gonorrhea, was generally susceptible to penicillin, until the emergence of resistant strains. Well-characterized genetic variations in the penicillin resistance-determining region correlate with decreased susceptibility to penicillin. At least 5 genes (penA, penB, mtrR, ponA, and penC) are involved in the chromosomally mediated resistance to this antibiotic. To date, no development of multiplex PCR assays targeting a range of gonococcal genes and variations as a means of predicting antibiotic resistance has been reported. The aim of this study was to develop a duplex assay using DNA from isolated strains. We describe the development and evaluation on the LightCycler platform of a real-time duplex PCR assay (hybridization probe format) for rapid and specific detection of ponA and penA variations, predicting penicillin susceptibilities. The real-time duplex PCR assay successfully detected variations in ponA and penA genes by use of distinct melting temperatures from a total of 120 Neisseria gonorrhoeae isolates. Moreover, the variation profiles obtained with the real-time PCR and the melting analysis showed good correlation with the pattern of penicillin susceptibility generated with classical antibiograms. Nucleotide sequencing data were in complete agreement with multiplex assay results. The presented assay is suitable for the detection of chromosomally mediated resistant strains of Neisseria gonorrhoeae in genotyping studies and could be valuable in the effective antimicrobial strategy to gonococci.

Development of multiplex polymerase chain reaction assay for simultaneous detection of clostero-, badna- and mandari-viruses along with huanglongbing bacterium in citrus trees

ABSTRACTMelissococcus plutonius is the causative agent of an important honeybee disease, European foulbrood (EFB). In addition to M. plutonius strains with typical characteristics (typical M. plutonius), we recently reported the presence of atypical M. plutonius, which are phenotypically and genetically distinguished from typical M. plutonius. Because typical and atypical M. plutonius may have different pathogenic mechanisms, differentiation of these two types is very important for diagnosis and more effective control of EFB. In this study, therefore, a duplex PCR assay was developed to detect and differentiate typical and atypical M. plutonius rapidly and easily. On the basis of the results of comparative genomic analyses, we selected Na+/H+ antiporter gene and Fur family transcriptional regulator gene as targets for detection of typical and atypical strains, respectively, by PCR. Under optimized conditions, the duplex PCR system using the designed primers successfully detected and differentiated all typical and atypical M. plutonius strain/isolates tested, while no product was generated from any other bacterial strains/isolates used in this study, including those isolated from healthy honeybee larval guts. Detection limits of the PCR were 50 copies of chromosome/reaction for both types, and it could detect typical and atypical M. plutonius directly from diseased honeybee larvae. Moreover, the duplex PCR diagnosed mixed infections with both M. plutonius types more precisely than standard culture methods. These results indicate that the duplex PCR assay developed in this study is extremely useful for precise diagnosis and epidemiological study of EFB.

Simplified extraction protocol of citrus tissues for rapid detection of ‘Candidatus Liberibacter asiaticus’ using isothermal recombinase polymerase amplification assay and its application for prevalence studies

Development of SCAR markers and PCR assays for single or simultaneous species-specific detection of Phytophthora nicotianae and Pythium helicoides in ebb-and-flow irrigated kalanchoe

Development and validation of a droplet digital PCR assay for the detection and quantification of Bartonella species within human clinical samples.

Specific scopeThis Standard describes a diagnostic protocol for ‘Candidatus Liberibacter africanus’, ‘Candidatus Liberibacter americanus’ and ‘Candidatus Liberibacter asiaticus’.1This Standard should be used in conjunction with PM 7/76 (5) Use of EPPO diagnostic protocols.Specific approval and amendmentFirst approved in 2014–09. Revised in 2021–04.