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Diarrhoeal Disorders

1. V. cholerae O1 and O139 serogroups are the causative agent of cholera leading to large morbidity and mortality.

2. Quick diagnosis of cholera is required for early treatment and control measures

3. Conventional methods of diagnosis is inconvenient and delayed process for early detection.

4. To overcome these difficulties, RMRC, Bhubaneswar has developed a Quadruplex PCR assay that can detect simultaneously serogroup, biotype, toxigenic potential and regulating factor, which is simple, rapid and cost effective.

5. Again to eliminate the complicated steps of Quadruplex PCR, we developed an easy PCR Kit that is very simple, rapid and cost effective and can be easily performed by trained technicians to give early diagnosis during cholera outbreaks.

1. CONVENTIONAL METHODS OF DIAGNOSIS FOR V. CHOLERAE

TCBS
Urease   TSI Manitol   Indole

2. PHOTOGRAPH OF QUADRUPLEX PCR ASSAY


Quadruplex PCR detected simultaneously serogroups (wbe/wbf), biotype ( tcpA, El Tor/Classical), toxigenic potential (ctxA) and regulating factor (toxR) of V. cholerae

3. PREPARATION OF AN EASY QUADRUPLEX PCR TEST KIT

Photograph of Quadruplex PCR Assay Using PCR Kit


Steps : Mastermix from ready made PCR Kit + DNA?PCR assay Electrophoresis Detection under UV

Quadruplex PCR assay of V. cholerae O1 using master mix from kit of 1 year old, preserved at -200C

CHOLERA OUT BREAKS IN ODISHA DUE TO V. CHOLERAE O1 EL TOR VARIANT

1. Cholera continues to be one of the major threats for public health problems particularly in developing countries caused by V. cholerae O1 and O139 serogroups.

2. Cholera has been reported in Odisha, India since last two decades caused by the dominant serogroup of V. cholerae O1 El Tor biotype till 2006

3. V. cholerae O1 El Tor variant harboring classical cholera toxin B gene caused a cholera epidemic in southern districts of Odisha in 2007.

4. Subsequently V. cholerae O1 El Tor variant spread to 26 districts during 2008-11causing cholera outbreaks.

5 Gradually El Tor variant is becoming dominant replacing the resident prototype El Tor vibrio.


Conventional methods of diagnosis for V. cholerae

New scheme for biotyping of V. cholerae O1

    Biotype    
Trait EL Tor Classical Hybrid El Tor Variant
VP test + - +/- +
Polymixin B(50U) - + +/- -
Chickencell aagglutination + - +/- +
ctxB genotype EL Tor Classical EL Tor/ Classical Classical

MAMA PCR assay V. cholerae O1 showing ctxB classical gene