2.2.2: i.e. 10x, 100x and 1000x from

2.2.2: qPCR Run 1

The
following ISO-certified SOPs for qPCR was followed and repeated for all 240
samples. The samples were divided into 5 batches, each containing 48 samples. Samples
were diluted 100x (using 495µl of NFW and 5µl of
extracted DNA samples) and two target-specific master-mixes were
prepared. The prepared master-mixes, NFW, diluted samples, pipette tips and
plates were loaded into the Hamilton Nimbus Liquid Handling machine. Upon completing
the automated pipetting, the 384-well plate was removed and 1010
standards for each target were manually serial diluted to 108, 106,
105, 104, 103, 102 and 101,
before being pipetted into the respective wells. The 384-well plate was then
sealed and centrifuged before conducting a qPCR run for two targets with QuantStudio
12K Flex. Results were exported to an Excel sheet for analysis with an Excel
macro-sheet. Any standard curve not fulfilling
the passing criteria of a R2 value above 0.99 would be re-ran with the same set of standards.
Samples which failed the macro-sheet passing criteria would be troubleshot –
samples with Ct (threshold cycle) values smaller than the standards’ starting
Ct value were too concentrated and would be re-diluted to 1000x; while samples
with Ct values larger than the standards’ ending Ct value have low DNA
concentration and would be re-diluted to 10x.

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2.2.3: qPCR Run 2 and 3

After
completing Run 1, the results will be complied into repeats, up to a maximum of
2 repeats. Samples would be re-diluted to their required dilution, i.e. 10x,
100x and 1000x from the extracted DNA samples. The presence of specific gut
microbiota was detected with qPCR. As different samples were repeated for the
12 targets, each 384-well plate would contain a single master-mix. The exported
results were analysed similarly as Run 1.

 

Other
general laboratory responsibilities included sending used glassware to Research
Support Centre (RSC) for autoclaving; laboratory housekeeping and
stock-keeping; monthly safety checks on eyewash stations, emergency showers,
fire extinguishers and first aid box in laboratory; and administrative work like
updating SDS (safety data sheets).

 

2.3: Training Received

Compulsory
laboratory related  inductions equipped me
with knowledge and understanding of the laboratory’s safety rules alongside
hazard reducing and reporting procedures Information provided regarding  whom to approach when laboratory stocks were
low and general laboratory information like storage location of required
laboratory items, allowed me to familiarize myself with the laboratory and
perform my duties independently,

SOP
(Standard Operating Procedure) trainings on qPCR and Standard Preparation
helped me perform my tasks confidently by clarifying my doubts, and teaching me
how to react to potential problems. Compulsory ISO (International Organization for
Standardization)-SOP multiple-choice questionnaire and checklist tests for qPCR
and Standard Preparation ensured I understood the protocols completely so that
I could perform the experiments independently and accurately.

            As quality is part of Danone’s culture, all Danone
members must undergo a FoQual (Focus on Quality) training, induction and yearly
quizzes. These FoQual inductions taught me the importance of quality of all
Danone’s functions, motivating me to play my role with responsibility.

Apart
from technical and compulsory trainings, I was also given the opportunity to
attend the Product Marketplace and Digital Marketplace events which exposed me
to Danone’s R&D contribution; future R&D plans; and products sold
worldwide. Danone’s trainings, events and VENUS project’s induction broadened
my understanding of the purpose behind the research I assisted in and motivated
me to perform all my tasks responsibly with quality to aid Danone in their
mission to bring health through food to as many people as possible.

 

 

 

2.4: Accomplishments

2.4.1:
Importance of Gut Microbiota

Human’s
intestinal tract contains hundred trillion bacteria (Savage, 1977). Healthy individuals’ commensal
microbiota can affect specific host developments and functions (Scholtens et al., 2011). Gut microbiota like Lactobacilli and Bifidobacterium
were associated with the immune system’s development and function (Souza et al., 2004) alongside preventing
pathogenic colonization (van der Waaji et al., 1971; Wells et al., 1988). Gut microbiota also aids in
biochemical pathways (Kovatcheva-Datchary et al., 2009), like vitamins
synthesis and metabolising complex proteins (Wall et al., 2009; Resta, 2009).

 

2.4.2: Factors Influencing Gut Microbiota

Microbial colonization supports
intestinal immune response development (Kelly et al., 2007; Gottrand, 2008) and begins immediately during and
after delivery. Infants’ gut microbiota is relatively dynamic in the first
years of life – its development relies mainly on diet and delivery mode (Scholtens et al.,
2011).

 

2.4.2.1: Delivery Mode

Delivery method affects infants’ gut
microbiota establishment. Neonates delivered vaginally is often colonized by
maternal perineal and vaginal microbiota, however Caesarian section (CS)-delivered
infants are commonly affected by nosocomial environment and skin. Vaginally
delivered neonates acquired microbiota resembling their mother’s vaginal
microbiota– mainly Sneathia, Prevotella and Lactobacilli species– while CS-delivered infants had microbiota
similar to their mother’s skin– namely Ropionibacterium
Corynebacterium and Staphylococcus species
(Dominguez-Bello et al., 2010).  Probiotic supplementation studies suggested a
strong correlation between infant colonization and maternal microbiota (Schultz et al, 2004; Gueimonde et al., 2006). However, the sole known prebiotic
supplementation study has shown that a significant increase in maternal
microbiota has no direct effect on the child’s intestinal microbiota (Shadid et al., 2007).

CS-delivered infants were colonized
less by Bacteroides and more by Clostridium difficile (Adlerberth et al.,
2007; Penders et al., 2006). CS-delivered infants also contain
less diverse and lower microbiota counts than vaginally-delivered infants (Biasucci et al., 2008; Gronlund et al., 1999). Additionally, Escherichia coli, Bacteroides and Bifidobacterium’s
growth is delayed in CS-delivered infants (Adlerberth
and Wold, 2009; Morelli, 2008; Biasucci et al.,
2008).

 

4.4.2.2: Type of Feeding

Facultative anaerobic bacteria – such
as Streptococci, are the initial
microbiota colonizing infants, reaching up to 108-1010cfu/g
of faeces within one to two days after birth (Mackie et al., 1999). Thereafter, Enterococcus–, Lactobacilli–,
and Staphylococcus–like species begin
colonization, generating a better condition for anaerobic bacteria (Orrhage and Nord, 1999).
After week one, Clostridium, Bifidobacterium, and Bacteroides are detected in infants’ faeces – with Bifidobacterium species dominating BM
(Breast Milk)-fed infants (Yoshioka et al., 1983). In addition to being a prebiotic
oligosaccharides source, BM also contains living bacteria, such as
Bifidobacterium, Streptococci, lactic
acid bacteria and Staphylococci (Collado et al.,
2010; Martin et
al., 2008; Perez et al., 2007).

Infants would be progressively
introduced to SWF (solid weaning food), such as fruits and cereals– introducing
soluble and insoluble non-digestible carbohydrates (NDCs), after drinking BM
containing only NDCs for about four to six months (Edwards and Parrett, 2002).  Studies show that Clostridium, Streptococci
and Bacteroides counts increased
after introducing SWF to BM-fed infants, while Bifidobacterium counts remained high and other anaerobes began
colonization (Stark and Lee, 1982). Roger et al. (2010)’s study show
that BM-fed infants had more diversified Bifidobacterium
population than formula-fed infants.

 

4.4.2.3: Antibiotic Usage

Usage of antibiotics may disrupt
biochemical pathways, leading to immune diseases such as eczema and IBD
(inflammatory bowel disease) (Kelly et al., 2007; Conroy et al.,
2009). Increased antibiotics usage would result in enhanced inflammatory
diseases (Greer and O’Keefe, 2011), with
exposure to antibiotics leading to decreased Bifidobacterium counts in infants’ gut and increased obesity risk
in adulthood (Martin and Walker, 2008; Penders et al.,
2006; Reinhardt et al., 2009).

 

2.4.3: VENUS Study

As BM enhances new-borns’ immune
system maturation (Biasucci et al., 2008), incorporation of probiotics and/or prebiotics
in infant formula have been attempted to develop formulas stimulating gut
colonization of BM-fed infants (Boehm and Moro,
2008; Puccio et al., 2007). Prebiotics increase SCFA (Short-Chained Fatty
Acids)’s production thus increasing host metabolism and mineral absorption (Saulnier et al., 2009)
alongside developing new-borns’ immune system, protecting them against
pathogens (Saulnier et al., 2009; Boehm and Moro,
2008).  Bifidobacterium growth is also selectively stimulated by synbiotics
(a combination of prebiotics and probiotics) (Parracho
et al., 2007; Puccio et al., 2007). VENUS is a randomized, controlled,
double-blind trial investigating the effects of a new infant formula in 725
healthy-term Singapore subjects on growth, body composition, tolerance and
safety.

 

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