Madiha Kiran (2008-VA-272)

Seroprevalence Investigations And Molecular Detection Of Mycoplasma Synoviae Using Loop-Mediated Isothermal Amplification (Lamp) In Poultry - 2016. - 53p.;

Pakistan poultry industry has been making remarkable progress from the last few decades but it is still threatened to certain infectious diseases which may cause heavy economic losses. Respiratory distress is of major concern, caused by a number of bacteria and viruses. Mycoplasma synoviae is the second most economically significant bacterial pathogen causing infectious sinusitis which contributes greatest in respiratory distress cases, and infectious synovitis. Serology is a best tool to detect the prevalence of infection in a flock but sometimes it is associated with imperfections in results. Accurate and early diagnosis through molecular technique is necessary for timely treatment of the disease. Loop-mediated isothermal amplification (LAMP) represents a rapid and sensitive alternative to conventional PCR, showing to be a robust, inexpensive, simple, and powerful method for selective and specific detection of M. synoviae.
A total of n=300 sera samples were collected from broiler and layer flocks. Samples were tested against MS antibodies by Rapid Serum Agglutination (RSA) using SPAFAS MS Plate antigen (Charles River Inc., CT, USA). For LAMP based identification, tracheal and synovial swabs samples were collected from sero-positive birds. To perform LAMP, DNA was extracted through organic extraction method from swab samples, as well as from Indicating FTA® classic cards (for positive control). The vlhA gene targeted primers were designed using Primer Explorer V5. Primers were synthesized over 200 nmol scale. FIP and BIP were subjected to Reverse Phase Cartridge (RPC) for purification. The LAMP reaction mixture was incubated at 60оC temperature in a water bath for 60 minutes. After amplification of DNA, Bsm DNA
polymerase had been inactivated at 80оC. Results were read after gel electrophoresis in 2% agarose gel. A real-time LAMP assay was also performed using ESEQuant tube scanner with FAM as reporting dye.
The total samples positive for MS infection were 153 when tested through RSA, indicating 51% seroprevalence of MS in poultry flocks of Jhang. The positive percentage for broiler and layer birds was 48% and 54%, respectively. LAMP assay detected 54% positive samples. The results of successful LAMP assay indicated a specific ladder-like pattern. The developed LAMP assay was found 100% specific, as it did not amplify the DNA of other mycoplasma species (M. gallisepticum and M. imitans). The sensitivity of the Real-time LAMP assay was noted as 10fg/μl of DNA.
The study concluded that Mycoplasma synoviae infection is prevalent in poultry flocks of District Jhang of Punjab, Pakistan, and the LAMP assay is a sensitive rapid molecular method for the detection of M. synoviae.
In summary, the developed LAMP assay is an ideal, robust and simple method to diagnose M. synoviae in poultry. Based on its high specificity and sensitivity, LAMP can be considered as a promising tool for small as well as large-scale detection of M. synoviae in poultry flocks.



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