sanjeev final multiplex pcr.docx

8/20/2019 sanjeev final MULTIPLEX PCR.docx

1/60

1. INTRODUCTION

Multiplex PCR is a techniques by which we can identify particular gene of any bacteria, any

cells or any type of organism by targeted their gene with with the specific primer, in this

technique we can use multiple of DNA and can amplify each DNA with multiple of primers at

one time to rapid identify multiple of organism from gien population of samples! "raditionally,

detection and enumeration of bacterial pathogen hae been largely based on the use of selectie

culture and standard biochemical method(Kong et al,).#ut these methods suffer from numbers

of drawbac$s% &irstly, Pathogenic bacteria which normally occurs in low numbers tend to incur

large number of error in sampling and enumeration (Fleisher JM). 'econdly, culture based

method are time consuming, tedious, inariably monospecific (i!e! detecting low output)!

"hirdly,many pathogenic organism in the enironment although iable, are either difficult to

culture or non%culturable (Fleisher JM), but can still cause illness (Rahman et al ,1996). Due to

this difficulties, examination of water samples for pathogens li$e vibrio cholerae, shigella

dysenteriae, aeromonas spp. and campylobacter spp.,etc! is normally not performed during

routine microbiological assesment of water quality (Kong et al,). "he aim of this study is to

screen different water bodies (tap water,pond water, rier water and fish water) for the presence

of ibrio species and determine their pathogenicity! "he $nowledge will help to control ibrio%

associated gastroentritis in india as the awareness of the danger associated with the consumption

of tap water, raw and undercoo$ed seafood will be created for the masses! Vibrio are gram%

negatie, straight,cured rod,non spore forming,motile,usually contain single polar flagellum

and halophilic,few species are non%halophilic those who are halophic they require salt

medium(Nacl) for their growth! Mostly are sensitie to acidic p*,and few are al$aline p*

tolerant! According to centre of disease control +, infection and - death happens in each

year by ibrio infection! "ill now date ibrio hae ./ species all around the world, out of ./

twele account for the ma0ority of ibrio infection in humans!ibrio are water surface organism

occurs in fresh water as well as salt water(rier,pond,fish and industrial effluent etc!!) they are pathogen of marine liing organism,this is the reason that doctor suggested do not to eat

uncoo$ed food (fish,lobster,crac$) becouse comsuming of uncoo$ed food is the indirect

pathway of ibrio to enter inside the human body!

1 | P a g e


2/60

V.cholerae,V.parahaemolyticus,V.vulnificus,V.alginolyticus,V.mimicus,V.fluvialis,V.adaptatus,V.c

ampbellii,V.diabolicus,V.fischeri,V.furnissii,V.tapetis V.hollisae, V.damsela and many more

species li$e this are human pathogen! Most of this species secrete enterotoxins in foods,water

and gastrointestinal tract! 1dentification of ibrio species is ery important for us becouse this

are the main cause of human infection! 1f we could identify that water bodies which are

contaminated by ibrio so that we can ma$e them free from pathogens and preent to cause any

water born disease again!

A multiplex polymerase chain reaction (PCR) method,specifically designed for application in

routine diagnostic laboratories, was deeloped for identifying 2 human pathogenic Vibrio

species: V.cholerae,V.parahaemolyticus,V.vulnificus,V.alginolyticus,V.mimicus! "his assay

directed toward the dna3 gene ( a house$eeping gene that encodes heat shoc$ protein 4,for the

identification of ibrio species) was tested on the total of 5+ strains representing /5 ibrio

species! 'pecific PCR fragments were formed in isolates which belong to the 2 target species

and were absent from all strains other than this 2 species, indicating high specificity of multiplex

PCR! "his technique represented a rapid detection of the 2 ma0or pathogenic ibrio species!

Most of the effort for ibrio identification were done with marine water, Now the present study

based on rapid identification of ibrio species form marine water as well as fresh water!

Color o! "i#rio s$e%ies %olonies on TC&' meia

Vibrio alginolyticus 6ellow

Vibrio vulnificus 7reen +28 or 6ellow 928

Vibrio cholera 6ellow

Vibrio mimicus 7reen

Vibrio parahaemolyticus #lue to 7reen centered colony

2 | P a g e


3/60

O#e%*i"es

9! 1solation and characteri:ation of ibrio species from tap water, pond water, fish water

and rier!

/! 1solation of DNA from the ibrio colonies obtained on the "'A media!5! ;ualitatie determination of the isolated DNA using Agarose 7el Double beam 'pectrophotometer!

2! "o confirm the pathogenic species by specific primer design from its toxin genes by

PCR!

-! 1dentification of human pathogenic ibrio species by multiplex PCR!

3 | P a g e


4/60

+. R-I OF /ITR0TUR

Oa2i et al.(+314)obsered that the regional differences in human papillomairus (*P>)

genotypes and the presence of mixed *P> infections may affect adersely the efficacy of the

*P> accine! "herefore, a simple and high%throughput *P> genotyping system is required!

Recently, a noel *P> genotyping $it (the Mebgen "M*P> $it) was deeloped! "his $it uses

multiplex PCR and ?uminex format! 1n the present study, the analytical performance of the

$itwas examined using *P> x MAP"M technology to detect 95 types of high%ris$ *P>s and an

internal control in a @-%well plasmid DNA! All 95 types of *P>s were detected with a minimum

detection sensitiity of /2 copiestest, and highly specific signals were obsered! *P> 9-

plasmid was detected in samples containing mixtures with other *P>%type plasmids in ratios

ranging from 9B9 to 9B9! No cross reactiity was obsered with DNA from /. types of other

infectious microbes! A clinical ealuation was carried out using cerical samples from 52-

patients with persistent abnormal smears diagnosed atmass public health screenings for cerical

cancer! "he samples were presered in "acas "M medium until analysis! *P> was detected in

9-/ (42!28) samples including 99 (-.!@8) with single infections and 2/(5/!98) with multiple

infections! "he type distribution of the 95 high%ris$ *P> was as followsB /+!48*P> 9-, 99!.8

*P> 9+, -!+8 *P> 59, 5!98 *P> 55, 5!.8 *P> 52, @!58 *P> 5@, 9!@8 *P> 42, +!-8 *P>

29,5.!8 *P> 2/, @!58 *P> 2-, 9-!.8 *P> 2+, 5!.8 *P> 2@, and 9!@8 *P> -+! "o

ealuate sample stability oer time, changes in the detection of *P> DNA deried from *e?a

and 'i*a cells were measured in 5 types of liquid%based cytology media! *P> DNA was

detected in "acas and "hinprep "M samples after storage at 4C or 5C for 4 wee$s and within

9 wee$ of collection in 'urepath "M samples! "hese results suggest that this newly deeloped

*P> genotyping $it is suitable for use in both clinical applications and large%scale

epidemiological studies!

5osies2il et al .(+314) described about the application of the PCR method for the

simultaneous detection of DNA of 7ram%negatie bacteria, 7ram%positie bacteria, yeast fungi

and filamentous fungi in blood and, thus, a whole range of microbial etiological agents that may

cause sepsis! Material for the study was sterile blood inoculated with four species of

microorganisms ( Escherichia coli, Staphylococcus aureus, Candida albicans and Aspergillus

4 | P a g e


5/60

fumigatus) and blood collected from patients with clinical symptoms of sepsis! "he deeloped

method is based on nested%multiplex real%time PCR !Analysis of the obtained data shows that

sensitiity of nested%multiplex real%time PCR remained at the leel of 99 C&=ml for each of

the four studied species of microorganisms and the percentage of positie results of the

examined blood samples from the patients was .8 and 9@8 for the microbiological culture

method! "he designed primers correctly typed the studied species as belonging to the groups of

7ram%positie bacteria, 7ram%negatie bacteria, yeast fungi, or filamentous fungi! Results

obtained by us indicated that the designed PCR methodsB (9) allow to detect bacteria in whole

blood samples, (/) are much more sensitie than culture method, (5) allow differentiation of the

main groups of microorganisms within a few hours!

Kama et al .(+314) reported that microscopy and antigen detecting rapid diagnostic tests are

the diagnostic tests of choice in management of clinical malaria! *oweer, due to their

limitations, the need to utili:e more sensitie methods such as real%time PCR (qPCR) is eident

as more studies are now utili:ing molecular methods in detection of malaria! 'ome of the

challenges that continue to limit the widespread utili:ation of qPCR include lac$ of assay

standardi:ation, assay ariability, ris$ of contamination, and the need for cold%chain!

?yophili:ation of molecular assays can oercome some of these limitations and potentially

enable widespread qPCR utili:ation! A recently published multiplex malaria qPCR assay was

lyophili:ed by free:ing drying into 'ample%Ready format (MM'R)! MM'R assay contained

all the required reagents for qPCR including primers and probes, requiring only the addition of

water and sample to perform qPCR! "he performance of the MM'R assay was compared to the

non%free:e dried, EwetF assay! 'tability studies were done by maintaining the MM'R assays at

four different ambient temperatures of 4GC, room temperature (R"), 5.GC and 4/GC oer a

period of 4/ days, tested at seen%day interals! Plasmodium falciparum and Plasmodium iax

DNAs were used for analysis of the MM'R assay either as single or mixed parasites, at two

different concentrations! "he C" alues and the standard deiations ('D) were used in the

analysis of the assay performance!"he limit of detection for the MM'R assay was !/44

parasitesH? for Plasmodium spp! (P?=) and P! falciparum (&A?) assay targets compared to

EwetF assay which was !5@ and 5!95 parasitesH? for P?= and &A? assay targets, respectiely!

"he MM'R assay performed with high efficiencies similar to those of the EwetF assay and was

5 | P a g e


6/60

stable at 5.GC for 4/ days, with estimated shelf%life of 2 months! Ihen used to analyse field

clinical samples, MM'R assay performed with 98 sensitiity and specificity compared to the

EwetF assay!"he MM'R assay has the same robust performance characteristics as the EwetF

assay and is highly stable! Aailability of MM'R assay allows flexibility and proides an option

in choosing assay for malaria diagnostics depending on the application, needs and budget!

5a*ier et al .(+314) obsered that :ymoseptoria tritici is a hemibiotrophic ascomycete fungus

causing leaf blotch of wheat that often decreases yield seerely! Populations of the fungus are

$nown to be highly dierse and poorly differentiated from each other! *oweer, a genotyping

tool is needed to address further questions in large collections of isolates, regarding regional

population structure, adaptation to anthropogenic selectie pressures, and dynamics of the

recently discoered accessory chromosomes! Procedure is limited by costly and time%consuming

simplex PCR genotyping! Recent deelopment of genomic approaches and of larger sets of

''Rs enabled the optimi:ation of microsatellite multiplexing!A reliable protocol to amplify /4

''Rs organi:ed in three multiplex panels, and coering all J! tritici chromosomes! Ie also

propose an automatic allele assignment procedure, which allows scoring alleles in a repeatable

manner across studies and laboratories! All together, tools enabled us to characteri:e local and

worldwide populations and to calculate diersity indexes consistent with results reported in the

literature!


7/60

house m%PCR! 'ingle%locus PCR reactions were performed and standardised, and then primers

for each locus in turn were added indiidually in subsequent amplifications until m%PCR was

achieed! Amplicons of the expected si:e were obtained from each of the four bacterial gene

fragments! &inally, the analytical specificity and limits of detection were tested! #ecause they

did not amplify any product from non%'"1 tested species, the primers were specific! "he

detection limits for the Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis

and reaplasma urealyticum primer sets were 2!9/ K 92, 5!@K95 , -9!9@ K 9- and -!5. K 92

copies of a DNA template,respectiely!"he methodology designed and standardised here could

be applied satisfactorily for the simultaneous or indiidual detection of Chlamydia trachomatis,

Neisseria gonorrhoeae, Mycoplasma hominis and reaplasma urealyticum! Method is at least

as efficient as other preiously described methodsL howeer, this method is more affordable for

low%income countries!

Wu et al .(+314) reported that the noel method to simultaneously detect expression of four

genes, ribonucleotide reductase subunit M9(RRM9), %ray repair cross%complementing gene 9

(RCC9), thymidylate synthase ("') and class 111 %tubulin ("=##5), and to assess their

application in the clinic for prediction of response of non%small cell lung cancer (N'C?C) to

chemoradiotherapy! Designed four gene molecular beacon (M#) probes for multiplex

quantitatie real%time polymerase chain reactions to examine RRM9, RCC9, "=##5 and "'

mRNA expression in paraffin%embedded specimens from 2 patients with adanced or

metastatic carcinomas! "wenty one N'C?C patients receiing cisplatin%based first%line

treatment were analy:ed! Molecular beacon probes could specially bind to their target genes in

homogeneous solutions! Patients with low RRM9 and RCC9 mRNA leels were found to hae

apparently higher response rates to chemoradiotherapy compared with those with high leels of

RRM9 and RCC9 expression (pO!2)! "he "' gene expression leel was not significantly

associated with chemotherapy response (p!2)!A method of simultaneously detecting four

molecular mar$ers was successfully established and applied for ealuation of

chemoradiotherapy response! 1t may be a useful tool in personali:ed cancer therapy!

Jeong et al .(+314) obsered that the wide range of inter%indiidual ariations in platelet

responses to clopidogrel! "he ariations in response to clopidogrel can be drien by genetic

polymorphisms inoled in the pathway of absorption, distribution, metabolism, excretion, and

7 | P a g e


8/60

the target receptor P/69/! A set of genetic ariants $nown for causing ariations in clopidogrel

responses was selected, which included C!"#C$%, &', &$( , C!"#)*&+, &* , &%, C!"'A+&$,

C!"'A-&', M/$ #*((0 1 A, '+'-C 1 , and "#!$# 2# ((+#1 C )! "he simultaneous

detection of these 9 ariants was deeloped by using a multiplex PCR and single%base

extension (M'') and hepatitis < irus

(*) are the ma0or causes of acute hepatitis worldwideL both *A> and * infection are a

main public health problem! A one%step multiplex reerse transcriptase quantitatie polymerase

chain reaction assay using hydrolysis probes was deeloped for simultaneously detecting *A>

and *! "his noel detection system proed specific to the target iruses, to be highly

sensitie and to be applicable to clinical sera samples, ma$ing it useful for rapid, accurate and

feasible identification of *A> and *!

Cora%h et al .(+314) reported that the microdeletions in the AJ& region of the 6 chromosome

are among the most frequent genetic causes of male infertility, although the specific role of the

genes located in this region is not fully understood! AJ&a and AJ&b deletions impair

spermatogenesis since no spermato:oa are found in the testis! Deletions of the AJ&c region,

despite being the most frequent in a:oospermic patients, do not correlate with spermatogenic

failure! "herefore, the aim of wor$ was to deelop a screening method to ascertain the presence

8 | P a g e


9/60

of the main spermatogenesis candidate genes located in the AJ&c region in the light of the

identification of those responsible for spermatogenic failure! DAJ, CD6, #P6/, PR6,

7?7A/?6 and C'7P4?6 genes were selected on the basis of their location in the AJ&c

region, testis%only expression, and confirmed or predicted protein codification! AM


10/60

were examined (.. animal nasal swabs, 99/ retail raw meat, and 42 deli meat)! "he multiplex

real%time PCR targeted the genesB nuc (identification of '! aureus), mecA (associated with

methicillin resistance) and P>? (irulence factor), and the primary and secondary enrichment

samples were assessed! "he conentional culturePCR method included the two%step selectie

enrichment, selectie plating, biochemical testing, and multiplex PCR for confirmation! "he

conentional culturePCR method recoered @2/54 positie '! aureus samples! Application of

real%time PCR on samples following primary and secondary enrichment detected '! aureus in

999/54 and 9//54 samples respectiely! &or detection of '! aureus, the $appa statistic was

!-+T!++ (from substantial to almost perfect agreement) and !/@T!.. (from fair to substantial

agreement) for primary and secondary enrichments, using real%time PCR! &or detection of mecA

gene, the $appa statistic was T!4@ (from no agreement beyond that expected by chance to

moderate agreement) for primary and secondary enrichment samples! "wo por$ samples were

mecA gene positie by all methods! "he real%time PCR assay detected the mecA gene in samples

that were negatie for '! aureus, but positie for 'taphylococcus spp! "he P>? gene was not

detected in any sample by the conentional culturePCR method or the real%time PCR assay!

Among '! aureus isolated by conentional culturePCR method, the sequence type '"5@+, and

multi%drug resistant strains were found in animals and raw meat samples! "he real%time PCR

assay may be recommended as a rapid method for detection of '! aureus and the mecA gene,

with further confirmation of methicillin%resistant '! aureus (MR'A) using the standard culture

method!

5imenes et al .(+314) obsered that the sexually transmitted diseases ('"Ds) may impair sperm

parameters and functions thereby promoting male infertility! "o date limited molecular studies

were conducted to ealuate the frequency and type of such infections in semen "hus, we aimed

at conceiing and alidating a multiplex PCR (M%PCR) assay for the simultaneous detection of

the following '"D pathogens in semenB Chlamydia trachomatis, Neisseria gonorrhoeae,

Mycoplasma genitalium, "richomonas aginalis, *erpes irus simplex (*'>) /9 and //, and

"reponema pallidumL Ie also inestigated the potential usefulness of this M%PCR assay in

screening programs for semen pathogens! 1n addition, we aimedB to detect human

Papillomairus (*P>) and genotypes by single PCR (sPCR) in the same semen samplesL to

determine the prealence of the seen '"Ds, *P> and co%infectionsL to assess the possibility

10 | P a g e


11/60

that these infections affect semen parameters and thus fertility! "he oerall alidation parameters

of M%PCR were extremely high including agreement (@@!/8), sensitiity (9!8), specificity

(@@!.8), positie (@-!48) and negatie predictie alues (9!8) and accuracy (@@!+8)!

"he prealence of '"Ds was ery high (22!58)! &urthermore, associations were obsered

between '"Ds and changes in semen parameters, highlighting the importance of '"D detection

in semen! "hus, this M%PCR assay has great potential for application in semen screening

programs for pathogens in infertility and '"D clinics and in sperm ban$s!

&orea et al .(+314) obsered that the among NQ cell receptor%ligand partnerships,

Q1R5D?9 and *?A%#w4 demonstrate the greatest diersityL permutations of their allelic

combinations titrate NQ reactiity! #alancing selection has maintained distinct subtypes of

Q1R5D?9 alleles in global populations, implying that each may proide unique fitness

adantages and ariably influence disease processes! "hough approaches exist for determining

*?A%# allotypes, practical methods for identifying Q1R5D?9 alleles are lac$ing! Ie hae

deeloped a PCR%based approach that identifies functional subtypes of Q1R5D?9 allelesL it is

suitable for research and may hae clinical application! 'ix allele subsets were identified based

on expression characteristics of the eleen most common Q1R5D?9 alleles represented in

reported populations! "he remaining -/ low%frequency alleles were distributed into these groups

based on sequence homology to coding regions! 'ubtype%specific 'NPs were found in exons 5,

4, and ., and used as priming sites for fie multiplex PCR reactions! 7enomic DNA deried

from 9.2 unrelated donors and 2/ related indiiduals from - families demonstrated !@@!28

concordance between sequence%based typing and our noel approach! &inally, PCR%based typing

accurately predicted NQ phenotypes obtained by flow cytometry after staining with D@ and

J/. monoclonal antibodies! "his noel approach facilitates high%throughput analysis of

Q1R5D?9 allotypes to enable a broader understanding of Q1R5D?9 and *?A%#w4 interaction

in health and disease!

Chng et al.(+314) was inestigate whether tissue samples processed by the rapid urease test

(R=") $it are suitable for dualpriming oligonucleotide%based multiplex polymerase chain

reaction (DP%PCR) to detect *elicobacter pylori (*! pylori )! A total of 24 patients with

specific gastrointestinal symptom were enrolled in this study! During endoscopy, gastric biopsy

11 | P a g e


12/60

specimens were ta$en for histology, R=", and DP%PCR! DP%PCR was performed on gastric

biopsy samples and tissue samples that were analy:ed by R=" at / separate institutes! 1n

detecting *! pylori , the concordance rate of the DPPCR tests between the tissue samples that

had been submitted to R=" and the gastric biopsy samples was inestigated! *! pylori co%

occurred with .-!8 (9@/2) of gastric ulcers, -4!58 (@94) of duodenal ulcers, and 55!58

(49/) of gastritis cases! *! pylori infection was found in 98 (55) of the patients with both

gastric and duodenal ulcers! erall, *! pylori was detected in 52 of 24 (-4!+8) patients! "he

diagnostic sensitiities of histology, R=", and DP%PCR were +2!.8 (552), .4!58 (/-52),

and @.!98 (5452), respectiely (P U !/)! "he positie predictie alue (PP>) of DP%PCR

was @4!48, whereas the negatie predictie alue (NP>) was @4!.8! 1n the rapid urease test

(C?test)%negatie cases, the frequency of positie DP%PCR and histologic results was /!8

(.52)! "he concordance rate of the DP%PCR tests between the tissue samples from the R="

$it and the gastric biopsy samples was @4!48 (2924)! "he rate of DPPCR and siler stain

positiity in the R="%negatie cases was /!8 (.52)!1n diagnosing *! pylori infection, DP%

PCR can be performed on tissue samples that hae been processed by the R=" $it! Particularly,

in patients with R="%negatie results, DP%PCR on these tissue samples could be helpful in

detecting of *! pylori infection.

et al .(+314) reported that the identification of human body fluids or tissues through mRNA%

based profiling is ery useful for forensic inestigations!Preious studies hae shown mRNA

biomar$ers are effectie to identify the origin of biological samples!'elected 9- tissue specific

biomar$ers to ealuate their specificities and sensitiities for human body fluids and tissues

identification, including porphobilinogen deaminase (P#7D), hemoglobin beta (*##) and

7lycophorin A (7?6) for circulatory blood, protamine / (PRM/) and transglutaminase 4

("7M4) for semen, mucin 4 (M=C4) and human beta defensin9(*#D9) for aginal secretion,

matrix metalloproteinases . and 99 (MMP. and MMP99) for menstrual blood, $eratin

4(QR"4)for oral mucosa, loricrin (?R) and cystatin - (C'"-) for s$in, histatin 5(*"N5) for

salia, statherin ('"A"*) for nasal secretion,dermcidin (DCD) for sweat and uromodulin

(=MD) for urine! "he aboe mentioned ten common forensic body fluids or tissues were used

in the ealuation! #ased on the ealuation, a reerse transcription (R") PCR multiplex assay,

C6R9, whichincludes 9/ biomar$ers (i!e!, *##, 7?6, *"N5, PRM/, QR"4, MMP99, M=C4,

12 | P a g e


13/60

DCD, =MD, MMP., "7M4, and '"A"*) and house$eeping genes Vi!e!, glyceraldehyde%5%

phosphate dehydrogenase (7APD*) and 9+'rRNAW, was deeloped!Assay was further alidated

with real casewor$ samples and moc$ samples (with both single source and mixture) and it was

approed that C6R9 is effectie to identify common body fluids or tissues (i!e!, circulatory

blood, salia, semen, aginal secretion, menstrual blood, oral mucosa, nasal secretion, sweat and

urine) in forensic casewor$ samples!

13 | P a g e


14/60

:! M0TRI0/ 0ND MT8OD'

:.1 Ma*erials

:.1.1 Reagen* Use

:.1.1.1 For Meia ;re$ara*ion

:.1.1.1.1 TC&' 0gar (Thiosl!a*e Ci*ra*e &ile 'al* '%rose 0gar)


15/60

DNA loading dye 2! Xl

Distilled water 9ml

:.1.1.4 Reagen* !or 0garose 5el le%*ro$horesis (1.+? 5el)

Agarose 9!/ gm

"A< buffer 2 (diluted to 9)


16/60

Reerse primer vibrio

parahaemolyticus

9Xl(+ppm)

"aq DNA polymerase 9Xl

Distilled water .!2Xl

DNA sample 2Xl

"otal /Xl

:.1.+ 5lassare Use

Conical &las$ Petri plate

Reagent #ottle #ea$er

7lass Rod ?%shaped rod

:.1.: Ma*erial ReBire

#utter paper 'patula Conical flas$

Cotton Aluminium foil Petridish

Micropipette Microtip 'preader

Clean film 1noculum loop 'pirit lamp

'pirit "est tube Measuring cylinder

"est tube stand Microcentrifuge tube ortex DNA sample

:.1.4 ReBire Ins*rmen*

16 | P a g e


17/60

ortex

Refrigerator 'pectrophotometer 7el doc

17 | P a g e


18/60

:.1.@ Colle%*e 'am$les< a*er sam$les (To*al < +6 'am$les)

:[email protected] Ta$ a*er sam$les

9! A0amgarh tap water(7ora$hpur)!

/! 3an$ipuram tap water(?uc$now)!

5! Qhurram Nagar tap water(?uc$now)!4! 7udamba "hana tap water(?uc$now)!

2! Cyto7ene tap water(?uc$now)!

-! #a$shi Qa "alab tap water(?uc$now)!.! &a0ulagan0 tap water(?uc$now)!

+! ##A= uniersity tap water(?uc$now)!

@! Adil Nagar tap water(?uc$now)!9Munshi Pulia tap water(?uc$now)!

:.1.@.+ ;on a*er

9! Dhatingra pond water(?uc$now)

/! 3an$ipuram pond water(?uc$now)!5! #sawan Purwa pond water(?uc$now)

4! Raitha pond 9st water(?uc$now)!

2! Raitha pond /nd water(?uc$now)!-! #a$shi Qa "alab pond water(?uc$now)!

:.1.@.: Ri"er a*er

9! 7omti rier(?uc$now)!

/! 6amuna rier(Delhi)!5! *indun rier(Delhi)!

4! 7anga rier(*aridwar)!

2! 7hagra rier(#araban$i)!

:[email protected] Fish a*er

9! Dhatingra fish water(?uc$now)!/! 3an$ipuram fish water(?uc$now)!

5! Munshi Pulia fish water(?uc$now)!

4! Qhurram Nagar fish water(?uc$now)2! "adi$hana fish water(?uc$now)!

18 | P a g e


19/60

:.1.6 ;rimer 'eBen%e !or Ml*i$le ;CR o! Fi"e ;a*hogeni% '$e%ies o! Vibrio

1able #: 3ist of "rimers used for amplification.

Targe* s$e%ies ;rimer 'eBen%e(@


20/60

:.+ MT8ODO/O5

:.+.1 Colle%*e 'am$les !or E$erimen*

"otal /- sample were collected for the purpose to done this experiment and each sample

collected from different areas! ut of /- mostly are collected from ?uc$now region and

some sample collected from different areas due to lac$ of rier in?luc$now / sample of

rier collected from Delhi,9 sample collected from #araban$i 9 from *aridwar and 9

sample of tap water collected from A0amgarh!

:.+.+ 'am$le ;ro%essing

After collection of the /2 sample the following sample processing was done are as

following methodsB

:.+.+.1 0*o%la"ing o! Meia

An autoclae is a deice used to strili:e equipment and supplies by sub0ecting them to

high pressure saturated steam at 9/9 GC for around 92T/ minutes depending on the si:e

of the load and the contents!

;ro%ere

"oo$ a labelled washed conical flas$!

Ieighed the media on weighing machine and then added in conical flas$ with distilled

water to ma$e total olume and stopped with cotton plug! Pac$ed petridish with paper and labelled them!

"ransferred labeled media (Date, type of media, name of person) to inside the autoclae

chamber! Closed the autoclae chamber!

Pressure reached at 92 lbs wait 92 minutes then stop the chamber!

"a$e out the sterili:ed media from autoclae for proceding next steps!

:.+.+.+ ;oring o! Meia In ;e*riish

Qept a media inside laminar flow then wash hand with spirit!

?abelled each plate( name,date,"ype of media,name of sample)!

Pour /2ml autocled media in each petriplate and leae it to solidify!

:.+.+.: '$reaing o! a*er 'am$le on TC&' Meia

20 | P a g e


21/60

"oo$ /Xl of water sample and put it on "C#' media!

'preader sterili:ed by dipping in alcohol and then by exposed with flame and then

allowed to cool!

'preaded all the inoculum eenly oer the surface of "C#' media!

Irapped plates with clean film then incubated at /+YC for / days and plates were

examined for bacterial growth! After incubation,petriplates were ta$en out from the incubator and bactrial colonies are

isible oer the surface of media!

:.+.+.4 '*rea2ing o! &a%*erial Colon=

'trea$ing is a technique used to isolate a pure strain from a single species of

microorganism, often bacteria! 'amples can then be ta$en from the resulting colonies

and a microbiological culture can be grown on a new plate so that the organism can be

identified, studied, or tested!

;ro%ere

Red hot inoculum loop and allowed the loop to cooled!

Pic$ed colony from "C#' media!

'trea$ the inoculm oer the surface of "'A media in a :ig :ag manner!

?abelled each plate with name,date,media,colony colour and sample name!

Irapped with clean film!

1ncubated at /+YC for 9 day!

:.+.+.@ Ino%la*ion o! 'ingle Colon= o! &a%*eria in Ts &ro*h

#roth is liquid neutrient medium which has all the component which is required for

bacterial growth,deelopment and diision!broth contained casein and soyabean proide

amino acids and nitrogenous source,glucose for energy source,nacl for maintaing

osmotic equilibrium, dipotassium phosphate acts as buffer to maintain p*!

;ro%ere

"oo$ autoclaed test tubes with labelled name,date,broth type and sample name!

Pour 9 ml autoclaed "' broth in each test tube with the help of sterili:ed measuring

cylinder! 1noculate single bacterial colony from "'A media to the "' broth!

21 | P a g e


22/60

Closed opening of test tube with cotton plugs and incubated at /+YC for 9 day inside the

incubator!

:.+.+.6 &a%*erial DN0 Isola*ion #= ;henol Chloro!orm Me*ho.

"he broth which was inoculated with bacteria and incubated for 9 day become the

sample for DNA isolation!

;ro%ere

&rom the liquid culture 9!2 ml of culture was ta$en in microcentrifuge tube and

centrifuge it at + rpm for 2 mins and pellet the bacterial cells! Resuspended the pellet in @Xl of the "< buffer!

Added 99 olume of 98 'D' solution(@Xl solution then add 9Xl of solution into

it)! Iaterbathed the tubes at 2%-YC and incubated it for approximately / hours!

After / hours the solution become thic$er then it wass in the starting,now mix -Xl of

PhenolBchloroformBisoamyl alcohol(/2B/4B9)to it and sha$e gently by repeatedly

inerting the tube! Ihite precipitate appears when mix this mixture is added to the tube and sha$ing

solution become creamy orange(white due to protein precipitation and orange due to

phenol)!

Qept for 2 min and then centrifuge at 9 rpm for 9 min! =pon centrifugation 5 layers appear in tube!

=pper aqueous layer in transparent li$e water which contain DNA below this is a thin

layer of white colour which is protein precipitate and the lower dar$ orange layer is

phenol chloroform mixture! Collected upper transparent aqueous layer in fresh new eppendorf tube and discard the

lower layer! Qept the eppendorf tube in refrigerator to cool it for 9%92 min!

Added double olume of chilled propanol to the aqueous DNA solution drop by drop

from its wall!after adding DNA precipitate out from the solution and $eep it again on YC

for 92 min,so that DNA is precipitated easily!

After precipitation centrifuge the tube at 9rpm for 9 min and discard the

supernatant,before discarding mar$ the DNA pellet with mar$er on wall of tube! Dried the DNA pellet in air or by $eeping the tube in inerted position on filter paper!

Dissoled the dry DNA pellet in 2Xl "< buffer!

Chec$ed the quality of DNA isolated by agarose gel electrophoresis!

22 | P a g e


23/60

:.+.+.A 7ali*a*i"e e*ermina*ion o! &a%*erial 5enomi% DN0 &= 0garose 5el

le%*ro$horesis (3.>?).

Dissoled agarose in !/4 gm in 5 ml "A< buffer in a conical flas$!

Placed the conical flas$ in an oen and heat until the solution becomes transparent!

=sed hand gloes to remoe the conical flas$ out from oen and wait until it becomes

pal bearable! Iiped the gel casting tray and comb with ethanol!seal the open 'ide using cello tape and

set up the apparatus! Poured the agarose solution in the casting tray!leae to solidify for 92%/ min!

After settling of the gel,mix 5Xl of gel loading dye with 2Xl of DNA sample with the

help of a micropipette! Carefully remoe the comb from the solidified gel so that the wells donSt brea$!

&ill the tan$ with "A< buffer!

Placed the agarose gel in the electrophoresis tan$!

"he well will be placed toward the cathode!

?oaded the sample in the gel carefully using pipettes!

Run the sample till it traels the half area of the gel!

Remoed the gel from the electrophoresis tan$ and obsere it on => transilluminator!

:.+.+.> '$e%*ro$ho*ome*ri% Ban*i!i%a*ion o! DN0

'pectrophotometry is the quantitatie measurement of the reflection or transmission

properties of a material as a function of waelength! spectrophotometry deals

with isible light, near%ultraiolet, and near%infrared! A spectrophotometer is

a photometer that can measure intensity as a function of the light source waelength! A

spectrophotometer is commonly used for the measurement of transmittance or

reflectance of solutions, transparent or opaque solids, such as polished glass, or gases!

*oweer they can also be designed to measure the diffusiity on any of the listed light

ranges that usually coer around / nm % /2 nm using different controls

and calibrations! Iithin these ranges of light, calibrations are needed on the machine

using standards that ary in type depending on the waelength of the photometric

determination!

;ro%ere

23 | P a g e


24/60

'witched N the => Double beam 'pectrophotometer and put the type of DNA to be

quantified! Prepare the dilution 9 Xl dsDNA sample and @@Xl ultrapure distilled water!

"hen $ept the sample in spectrophotometer and ta$e the reading of sample at /-/+

nm! Prepare a $nown dilution of DNA sample in "< buffer which was used to dissole the

DNA sample! Recorded the D of the sample at /- and /+ nm!

Calculated the concentration of the DNA from the obsered reading of absorbency!

Absorption ratioU/-/+ nm waelength

A/-/+U9!.%/! (9!+)

A%/-%Absorption by DNA

A%/+%Absorption by protein

:.+.+.9 ;ol=merase Chain Rea%*ion

PCR is a rapid technique for in itro amplification of a specific DNA fragment by use

of two short single stranded primer flan$ing this segment!PCR were used to amplify the

desired region of the gene!the PCR reaction was done in a sterile !2 ml tube,the

reaction mix was made in the following order!

:.+.+.9.1 ;rea$ra*ion o! ;CR MiE*re

1able ': /eagent for master mi4 preparation.

Chemi%al '*o%2 or2ing

PCR buffer 9x /Xl(9x)

dN"P /!2mM /!2Xl(!/mM?)

primer 9ppm

uniersal forward primer 9Xl(+ppm)

24 | P a g e


25/60

Reerse primer vibrio alginolyticus 9Xl(+ppm)

Reerse primer vibrio vunificus 9Xl(+ppm)

Reerse primer vibrio cholera 9Xl(+ppm)

Reerse primer vibrio mimicus 9Xl(+ppm)

Reerse primer vibrio parahaemolyticus 9Xl(+ppm)

"aq DNA polymerase 9XlDistilled water .!2Xl

DNA sample 2Xl

"otal /Xl

;ro%ere

Mixed the reagent !2 ml tubes or the !/ ml PCR tubes!

#e sure to $eep the reagent on ice !

>ortex the tube so that all the composition mix properly! Placed all the tube inside the thermocycler!

:.+.+.9.+ ;CR Coni*ion

1able +: /e5uired condition for "C/ amplification

'*e$s Tem$era*re ReBire

*eating lid 92YC1nitial denaturation @4YC for 5 min

Denaturation @4YC for 5sec

Annealing -YC for 5 sec

for 42 min on a

9!/8 gel!

25 | P a g e


26/60

4. R'U/T 0ND DI'CU''ION

4.1. E$erimen*al sam$le

Ie done this experiment by ta$ing /- different water sample from different area of

?uc$now region specially and some sample from other regions(A0amgarh tap water sample

from 7ora$hpur region,6amuna and *indun rier water from Delhi,7hagra rier sample

from #araban$i)!9 sample of tap water,- sample pond water,2 sample of rier water,2

sample of fish water!

"he gien sample collected for this study

fig +.$.$6 1ap 7ater samples6 83eft to /ight9 Aamgarh,;an


27/60

fig +.$.#6"ond 7ater samples6 83eft to /ight9hatingra,;an


28/60

samples too$ /Xl aliquots of each sample and spread it with glass spreader on each of the

labeled "C#' media! 1ncubated these plates at 5YC for ./ hours!

4.+.1 Ta$ a*er s$reaing

>ig +.#.$.$6 1C)S media

28 | P a g e


29/60

>ig+.#.$.#:1C)S media?Adil Nagar tap7ater spreaded on 1C)S media? yello7 colonies

obtained?sucrose positive.

"he yellow colonies are grown at corner wall of "C#' spreaded petriplate after incubation of 4+

hours!"he yellow colonies shows the sucrose positie!

>ig+.#.$.':1C)S media? Aamgarh tap 7ater spreaded on 1C)S media? no colonies obtained.

No colony grown in A0amgarh water sample!

29 | P a g e


30/60

>ig+.#.$.+:1C)S media?)aig+.#.$.*:1C)S media?Cyto0ene tap7ater spreaded on 1C)S media? yello7 and green

colonies obtained?sucrose positive8!9 and sucrose negative809.

30 | P a g e


31/60

?arge yellow and green pointed colonies are grown on "C#' spreded petriplate after incubation

of 4+ hours!"he yellow colonies shows the sucrose positie and green shows sucrose negatie!

>ig+.#.$.(:1C)S media?>aulagan tap7ater spreaded on 1C)S media? no colonies obtained.

No colony grown on &a0ulagan0 water sample!

>ig+.#.$.:1C)S media?0udamba 1hana tap 7ater spreaded on 1C)S media? no colonies

obtained.

No colonies grown in 7udamba Qa "hana water sample!

31 | P a g e


32/60

>ig+.#.$.%:1C)S media?;an


33/60

>ig+.#.$.$$:1C)S media?Munshi "ulia tap7ater spreaded on 1C)S media? yello7 and green


'mall and large spreded form of green and yellow colonies grown on "C#' spreaded petriplate

after incubation of 4+ hours!"he yellow colonies show the sucrose positie and green show the

sucrose negatie!

4.+.+ ;on a*er s$reaing

>ig+.#.#.$:1C)S media?)a


34/60

>ig+.#.#.#:1C)S media?)sa7an "ur7a pond 7ater spreaded on 1C)S media? yello7 colonies


'mall dispersed and large pointed yellow colonies grown on "C#' spread petriplate after /4

hours! "he yellow colonies show the sucrose positie!

>ig+.#.#.':1C)S media?hatingra pond 7ater spreaded on 1C)S media? yello7 and green


'mall green and yellow colonies are grown on "C#' spread petriplate after incubation of /4

hours! "he yellow colonies show the sucrose positie and green show the sucrose negatie!

34 | P a g e


35/60

>ig+.#.#.+:1C)S media?;an


36/60

>ig+.#.#.*:1C)S media?/aitha pond 7ater spreaded on 1C)S media? yello7 colonies


?arge and small dispersed yellow are grown on "C#' spread petriplate after incubation of /4

hours! "he yellow colonies show the sucrose positie!

4.+.: Ri"er a*er s$reaing

>ig+.#.'.$:1C)S media?0anga river 7ater spreaded on 1C)S media? 0reen colonies obtained?

sucrose negative.

'mall green colonies grown on "C#' spread petriplates after incubation of /4 hours!"he green

colonies show the sucrose negatie!

36 | P a g e


37/60

>ig+.#.'.#:1C)S media?0hagra river 7ater spreaded on 1C)S media? yello7 and 0reen

colonies obtained? sucrose positive and sucrose negative.

'mall green and large yellow colonies are grown on "C#' spread petriplate after incubation of

/4 hours! "he yellow colonies show the sucrose positie and green show the sucrose negatie!

>ig+.#.'.':1C)S media?0omti river 7ater spreaded on 1C)S media?!ello7 and 0reen

colonies obtained? sucrose positive8!9 and sucrose negative809.

?arge,small yellow and dispersed green colonies grown on "C#' media spread petriplate after

incubation of /4 hours! "he yellow colonies show the sucrose positie and green show the

sucrose negatie!

37 | P a g e


38/60

>ig+.#.'.+:1C)S media?!amuna river 7ater spreaded on 1C)S media? !ello7 colonies

obtained? sucrose positive.

'mall yellow dispersed colonies are grown on "C#' media spread petriplates after incubation

of /4 hours! "he yellow colonies show the sucrose positie!

>ig+.#.'.-:1C)S media?2indun river 7ater spreaded on 1C)S media? !ello7 colonies


?arge yellow colonies are grown on "C#' media spread petriplates after incubation of /4 hours!

"he yellow colonies show the sucrose positie!

38 | P a g e


39/60

4.+.4 Fish a*er s$reaing

>ig+.#.+.$:1C)S media?hatingra fish 7ater spreaded on 1C)S media? yello7 colonies


?arge,'mall dispersed yellow colonies are grown on "C#' media spread petriplates after

incubation of /4 hours! "he yellow colonies show the sucrose positie!

>ig+.#.+.#:1C)S media?;an


40/60

>ig+.#.+.':1C)S media?=hurram Nagar fish 7ater spreaded on 1C)S media? yello7 colonies


'mall yellow colonies are grown on "C#' media spread petriplates after incubation of /4 hours!

"he yellow colonies show the sucrose positie!

>ig+.#.+.+:1C)S media?Munshi "ulia fish 7ater spreaded on 1C)S media?!ello7 and 0reencolonies obtained? sucrose positive8!9 and sucrose negative809.

'mall,large green and small,large yellow colonies are grown on "C#' media spread petriplate

after /4 hours! "he yellow colonies show the sucrose positie and green show the sucrose

negatie!

40 | P a g e


41/60

>ig+.#.+.-:1C)S media?1adi


42/60

>ig +.'.#:1SA media?cytogene tap 7ater streaig +.'.':1SA media?Adil Nagar,))A,Munshi "ulia tap 7ater 80reen and yello79 colony

streaig +.'.+:1SA media?;n


43/60

>ig +.'.-:1SA media?)sa7an "ur7a pond 7ater8!9,hatingra pond 7ater8!9,/aitha pond

7ater8!9,an


44/60

>ig +.'.:1SA media?hatingra pond 7ater809 streaig +.'.%:1SA media?1adi


45/60

4.4 &ro*h %l*re

&or DNA isolation we required a liquid medium of bacteria so that i created a liquid

suspension culture of bacteria("' broth)!

>ig +.+.$: 1S )roth inoculated from 1SA media8left to right96))A tap 7ater809,Adil Nagar

tap 7ater8y9,Munshi "ulia tap 7ater80 !9,hatingra pond 7ater80 !9,;an


46/60

&ig 4!4!5B 1S )roth inoculated from 1SA media8left to right96Munshhi "ulia fish 7ater80

!9,1adiig +.+.+: 1S )roth inoculated from 1SA media8left to right96;naig +.-.$ :83eft to /ight9Bsolated NA and reagent used in NA isolation6$ 1E buffer,$@D

SS, phenol:chloroform:isoamyl alcohol,"ropanol.

46 | P a g e


47/60

4.6 0garose 5el le%*ro$horesis(3.>?)

&or the confirmation of DNA presence !+ 8 gel was prepared in order to isulai:e DNA under

=> transilluminator!

>ig +.*.$: /eagent used for Agarose gel formation6$ 1AE buffer and Agarose.

>ig +.*.#:6 @.D Agarose gel visualied in 0el documentation System. Lane 1: ladder 2:

;an


48/60

>ig +.*.':6 @.D Agarose gel visualied in 0el documentation System. Lane 1: ladder 2:B sa7an pur7a 3: /aitha pond 4: )a


49/60

4.A.+ ;on a*er

1able *: Spectrophotometric analysis of NA 8"ond 7ater9

'!No Pond water samples M factor Ratio DNA Conc Protein Conc

9 Dhatingra 7 9! 9!/ /!+ -!5.

/ Dhatingra 6 9! 9!9 9!++ 4!5-

5 3an$ipuram 6 9! 9!- .!5 94!+/

4 #sawan Purwa 9! !@. !@5 /!59

2 Raitha Pond 9! 9!5 4!5 @!2

- Raitha Pond 9! 9!/ /!/ 2!9

. #a$shi Qa "alab 9! 9!/ 4!/2 @!--

4.A.: Ri"er a*er

1able (: Spectrophotometric analysis of NA 8river 7ater9

'!No Rier water

samples

M factor Ratio DNA Conc Protein Conc

9 7omti 7 9! 9!@ -!-9 95!//

/ 7omti 6 9! 9!4 2!52 99!-@

5 6amuna 9! 9!4 2!+@ 95!9

4 *indun 9! !@@ 9!@/ 4!2-

2 7anga 9! !@@ /!. 4!@+

- 7hangra 7 9! 9! 9!25 5!2+

. 7hangra 6 9! 9! 9!-. 5!@

4.A.4 Fish a*er

1able : Spectrophotometric analysis of NA8fish 7ater9

49 | P a g e


50/60

'!No &ish water

samples

M factor Ratio DNA Conc Protein Conc

9 Dhatingra 9! 9!/ /!.+ 2!/.

/ 3an$ipuram 9! 9!/ /!// 2!/

5 Munshi Pulia 7 9! 9!/ /!@/ 2!2.4 Munshi Pulia 6 9! 9!/ 9!5+ /!@.

2 Qhurram Nagar 9! 9!/ /!/4 2!+

- "adi$hana 7 9! !@+ !2. 9!5@

. "adi$hana 6 9! 9!9 9!@. 4!22

4.> Ml*i$leE ;CR

&or confirmation of bacteria which is resposible for contaminating each water sample using

5 primers with each bacteria! "he bacteria which gie yellow colour on "C#' media those

DNA amplified with vibrio alginolyticus, vibrio vulnificus, Vibrio cholera! And those who

gie green colour on "C#' those DNA amplified with vibrio vulnificus, vibrio mimicus and

vibrio parahaemolyticus!

4.9 0garose gel ele%*ro$horesis (1.+?)

"he amplified DNA run on 9!/8 for the confirmation of which primer bind to the bacterial

DNA!

50 | P a g e

1 2 3 4 5 6 7

8 9 10


51/60

>ig +.%.$ Amplified NA using specific primers run on a $.# D Agarose gel

lane 1: marig +.%.# Amplified NA using specific primers run on a $.# D Agarose gel

lane 1: mar


52/60

>ig +.%.' Amplified NA using specific primers run on a $.# D Agarose gel lane 1: marig +.%.+ Amplified NA using specific primers run on a $.# D Agarose gel

lane 2: mar


53/60

>ig +.%.- Amplified NA using specific primers run on a $.# D Agarose gel

lane 2: mar


54/60

4.13 Ta#le o! vibrio s$e%ies $resen* in gi"en a*er sam$les

1able %: Amplification observed in different 7ater samples 7ith V.cholerae, V.parahaemolyticus,

V.vulnificus, V.alginolyticus, V.mimicus

'.NO 0TR '0M;/' CO/ON

-.0 -.-

(=ello)

-.-

(green)

-.C -.M -.;

9! A0amgarh tap water %%%%%%%%

/! 3an$ipuram tap water 7 ✓

5! Qhurram nagar tap water 6 ✓ ✓

4! 7udamba thana tap water %%%%%%%%%

2! Cytogene tap water 7,6 ✓

-! #a$shi $a talab tap water %%%%%%%%%

.! &a0ulagan0 tap water %%%%%%%%%

+! ##A= tap water 7 ✓

@! Adil nagar tap water 6 ✓

9! Munshi pulia tap water 7,6 ✓

99! Dhatingra pond water 7,6 ✓ ✓

9/! 3an$ipuram pond water 6 ✓

95! #sawan purwa pond water 6 ✓

94! Raitha pond 9 pond water 6 ✓

92! Raitha pond / pond water 6 ✓

9-! #a$shi $a talab pond water 7 ✓

9.! 7omti rier 7,6 ✓ ✓ ✓

9+! 6amuna rier 6 ✓

9@! *indun rier 6 ✓ ✓

/! 7anga rier 7

/9! 7hagra rier 7,6 ✓

//! Dhatingra fish water 6 ✓ ✓

/5! 3an$ipuram fish water 6

/4! Munshi pulia fish water 7,6 ✓ ✓ ✓

/2! Qhurram nagar fish water 6 ✓

/-! "adi$hana fish water 7,6 ✓

ello %olon= #a%*eria U >!A, >!>, >!C

5reen %olon= #a%*eria U>!>, >!M, >!P

Vibrio alginolyticus U yellow colony

Vibrio vulnificus U green +28 or yellow 928 colony

54 | P a g e


55/60

Vibrio cholera Uyellow colony

Vbrio mimicus U green colony

Vibrio parahaemolyticus U green centered colony

55 | P a g e


56/60

@. CONC/U'ION

"he samples were collected in order to detect presence of human pathogenic vibrio species in

water bodies and the presence of vibrio species was confirmed by spreading water sample on

"C#' media, incubated for 4+ hours at 5.YC resulting in many different colonies! 1t was

sub0ected to strea$ing on "'A media and incubated it for /4 hours (5.YC) followed by

inoculating it in "' broth, incubated for /4 hours (5.YC)! DNA was isolated from each sample

by phenolBchloroform method and the presence of specific vibrio species in water samples

detected by Multiplex PCR amplification method with different primers of vibrio species

(V.cholerae, V.parahaemolyticus, V.vulnificus, V.alginolyticus, V.mimicus9."he Amplified

product was analysed by electrophoresis, it shows the presence of particular human pathogenic

vibrio species in water samples! Among the tap water sample railway station (A0amgarh),

7udamba "hana (?uc$now), #a$shi $a "alab (?uc$now), &a0ulagan0 (?uc$now) were free from

vibrio as absence of amplified band showed but the sample of 3an$ipuram (?uc$now) showed

the presence of vibrio parahaemolyticus, sample of Qhurram Nagar showed the presence of

vibrio vulnificus and vibrio cholera, sample of Polytechnic chauraha showed the presence of

vibrio vulnificus, sample of ##A= showed the presence of vibrio mimicus, sample of Adil

Nagar showed the presence of vibrio vulnificus and sample of Munshi Pulia showed the

presence of vibrio parahaemolyticus! Among the pond water sample Dhatingra showed the

presence of V.vulnificus V. parahaemolyticus, sample of 3an$ipuram showed the presence of

V.vulnificus, sample of #sawan Purwa showed the presence of V. alginolyticus,sample of #a$shi

$a "alab showed the presence of V.parahaemolyticus,sample of Raitha pond 9st and /nd water

showed the presence of V.cholerae.Among the Rier water sample 7omti rier showed the

presence of vibrio vulnificus, cholera Z parahaemolyticus, sample of 6amuna rier showed the

presence of vibrio vulnificus, sample of *indun rier showed the presence of vibrio

alginolyticus Z vibrio vulnificus, sample of 7hagra showed the presence of vibrio

parahaemolyticus! All samples showed the presence of specific primers except 7anga Rier!Among the &ish water sample Dhatingra fish water showed the presence of V.alginolyticus Z

V.cholerae, sample of Munshi Pulia showed the presence of V.vulnificus Z

V.parahaemolyticus,sample of Qhurram Nagar showed the presence of V.cholerae,sample of

"adi$hana fish water showed the presence of V.parahaemolyticus. All fish water showed the

presence of vibrio species except 3an$ipuram fish water!

56 | P a g e


57/60

6.RFRNC'

5a*ier,0., Mar%el,T., Con!ais,J., Crane,C., Kema,5., '!!er*,F., an al2er,0iruses 1nfecting ?aboratory Rodents! P?o' N< @(2)B

[email protected]/2!

57 | P a g e


58/60

Jeong ,8., /ee,'


59/60

-elas%o,-., 'heroo,J'., Roas


60/60

page no 9.,/.%4@,2/%24 on A4 coloured %%%5 copies on A4 for spiral and rest #I

page no 9.,/.%4@,2/%24 coloured on butter paper 9 copies for hard bind rest paper #I

sanjeev final multiplex pcr.docx

Documents