FILARIASIS DETECTION OF VECTOR MOSQUITO VIRUS BY

USING PCR (polymerase chain reaction)

PROPOSED PRACTICE FIELD WORK

By
Fikrie Fauzan
BIA015086

MINISTRY OF RESEARCH, TECHNOLOGY AND HIGHER EDUCATION

UnSoed
FACULTY OF BIOLOGY
PURWOKERTO

2018
FILARIASIS DETECTION OF MOSQUITO VECTOR USING PCR (polymerase
chain reaction)

FIKRIE Fauzan
BIA015086

To meet the requirements obtained his Bachelor of Science at the Faculty of Biology,
University of General Sudirman Purwokerto

Approved and endorsed

at the date of,

Supervisor, Field Supervisor,

Dr. Daniel Joko Wahyono, M. Biomed Graduated Susanti, S.KM., M.PH

NIP.19630913199103 1003 NIP. 19801206200604 2003

Knowing,
Vice Dean for Academic Affairs of the Faculty of Biology
UnSoed

Dr. Hendro Pramono, MS.

NIP. 19590722198601 1001

i
 Preface

Praise Allah SWT writer who has given His grace and guidance so that I can finish
the Job Training Report. Job Training report entitled "Detection of Filariasis From
Mosquito Vector Method Using PCR (Polymerase Chain Reaction) ",held on January
17 to February 6, 2018 at the Great Hall of other research and development Disease
Vector and Reservoir (B2P2VRP) Salatiga.
The author realizes that in the preparation of the Work Plan Field can not be solved
without the help of various parties. Therefore, on this occasion the author would like to
thank the parents who have provided moral and material support, Dr. Hendro Pramono,
MS As Vice Dean for Academic Affairs of the Faculty of Biology who has memberiakn
permission to carry out Field Work Practice, Mr. Joko Waluyo, BSc, ST, Dipl.EIA,
M.Sc.PH as head B2P2VRP that has granted permission Field Work Practice. Dr. Daniel
Joko Wahyono, M. Biomed, which has been guiding and assisting the process of
preparation of the street vendors. Mother Passed Susanti, S.KM., M.PH as guides Job
Training in Research and Development of Disease Vector and Reservoir Salatiga. Dra.
Hernayanti M, Si.
The authors recognize that writing of this proposal is far from perfect. For that all
criticism and constructive suggestions for the perfection ditulisan is expected to come. I
hope this proposal can be useful for writers in particular and readers in general.

Purwokerto, February 2018

Author

ii
 TABLE OF CONTENTS

Page
foreword ................................................................................................................ ii
I. Introduction ...................................................................................................... 1
II. Material and Methods ...................................................................................... 3
III. Working draft .................................................................................................. 7
Reference list ........................................................................................................ 9

iii
 I. Introduction

A. Background

Filariasis (elephantiasis) is a chronic infectious disease caused by a filarial worm

which is transmitted through mosquito vectors. Adult filarial worms in humans living in
the tract and lymph nodes that can cause acute clinical symptoms such as inflammation
of lymph nodes and channels. Inflammation can develop into lymphoedema and
hydrocele persistent and intractable, and can cause permanent disability (Santoso &
Nungki, 2015).
In the world 90% of cases are caused by Wucheria bancrofti filariasis followed
Brugi malayi and Brugi timori (DAS et al., 2002). An estimated 23 species of mosquitoes
of the genus Anopheles, Aedes, Culex and Mansonia can support the development and
brugian bancroftian filariasis (Schmidt and Robert, 2000). According to Yahya et al.
(2014), generally Wucheria bancrofti transmitted by various types of mosquito Culex sp.,
Anopheles spp., And Aedes sp. Filarial development in the mosquito takes approximately
2 weeks. The life cycle begins when a mosquito as a vector bite and suck the blood of
infected people active filariasis. In the time since the bancroftian filariasis infection to
appear microfilariae in the blood is approximately 6-12 months of being on filariasis
Brugian approximately 3.5 months (MOH, 2002).
Based on the results of the 2000 survey, the number of chronic filariasis patients
were reported as 6,223 people spread over 1,553 villages in 231 dabupaten, 26 provinces.
These data are not depict the actual data because it was reported by 42% of the 7,221
health centers. Filariasis endemicity level in Indonesia based on survey results of 1999
finger blood is still high with an average of microfilariae (mf rate) of 3.1% (from 0.5 to
19.64%). Based on the survey in 2002-2005 found that the amount of patients occurred
on that time frame, especially in Sumatra and Kalimantan. With mf rate of 1% or more in
84 districts identified (DITJEN PPM and PL 2005). The clinical symptoms are very
diverse (variation), ranging from no symptoms (asymptomatic) to severe. It depends on
the geographic area, the species of parasite, responsiveness (response) immune patient,
and the intensity of infection. Symptoms usually appear after 3 months of infected
(infection), but most of the incubation period of between 8-12 months (Malewa, 2008).
Mosquitoes of the genus Culex mosquitoes are abundant around us. Culex
mosquitoes includes insects that have proven to be a vector of various diseases. Culex

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quiquefasciatus is one of the genus Culex species that can act as vectors of filariasis.
Generally genus Culex like dumpsites households. Culex sp. has a habit of laying eggs in
clusters on the surface of the water. The eggs will hatch into larvae within 2-3 days. Culex
sp. has 4 times the growth (instar I-IV) and becomes a pupa within a period of 8-14 days,
the pupa will be eggs within 1-2 days (Sholichah, 2009).
One of the methods used for the detection of filariasis is by using Polymerase
Chain Reaction (PCR). PCR method used is the method of Chelex 100. The PCR test
steps include preparation, DNA isolation, Running PCR and electrophoresis readout of
results. Isolation is done by separating the mosquito's body taken part proboscis. Stages
followed by running PCR that include denaturation, annealing and extension. Results of
running the PCR and then do the reading results of the electrophoresis method in the
media sample agarose gel, a positive result when the band formed in gel media (Santoso
& Nungki., 2015).
Aim
Objective job training at the Center for Research and Development of Disease
Vector and Reservoir (B2P2VRP) Salatiga is to:
1. Gain experience of working environment, ethics, and competencies required
in the workplace.
2. Capable of detecting filariasis in the mosquito vector by using Polymerase
Chain Reaction (PCR).

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 II. MATERIALS AND METHODS OF WORK

A. Materials

Tools used include a micropipette, microtube rack, clean bench, microsentrifuge,

1.5 ml microtube, microtube PCR, pipette tips, PCR machines, incubators, vortex, stereo
microscope, freezer, pestle, gridder, mini-coloumn, microtube rack 1 , 5 ml, PCR rack,
paper cups, gauze, markers or marker, microwave, mold agar, thick glass bottles, beakers,
computer and pitting electrophoresis.
Materials used include mosquitoes, silica gel, 70% alcohol, a DNA extraction kit
(DNeasy), distilled water, PBS 1x, digestion K, protein kinase C, RNase solution, lysis
of binding buffer, ethanol 96%, wash buffer I, wash buffer second, the elution buffer,
master mix, primer Hha I (working solution 10x), ddH2O, isolate DNA (aliquote), the
control, the solution TAE / TBE 1x, marker, Syber safe, agarose, pcr products, masks and
gloves.

B. Location and time Field Work Practice

Field Work Practice was held on 17 January-February 6th, 2018 in Molecular
Laboratory, Center for Research and Development of Disease Vector and Reservoir
(B2P2VRP), Salatiga.

C. Ways of working

1. Extraction (Laboratory of Molecular, 2017).

1.1, Sample obtained is checked based on field data.

1.2, Sample inserted into the tube or pials with the maximum number of 25
animals each tube
1.3. A total of 180 uLbuffer ATL is added to each Pials.
1.4. A total of 20 uL Proteinase K added to Pials.
1.5. Every Pials mashed with in milled using a pellet pestle, then divortex and
incubated overnight at 37 ° C.
1.6. The results of the incubation and then moved to coloumn mini tube with a
collection tube, then added 200 uL Buffer AL.
1.7. Collection tube replaced, 2 ml DNeasy kit added to the tube, centrifuged for
1 minute at a speed of 6000 rpm.

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 1.8. Collection tube replaced, as many as 500 uL AW 1 (Wash Buffer) is added
to the mini coloumn tube, centrifuged for one minute at a speed of 6000 g
(8000 rpm).
1.9. Collection tube replaced, as many as 500 uL AW 2 (Wash Buffer) is added
to the mini coloumn tube, centrifuged selama3 minutes at 20,000 g (14,000
rpm).
1:10. Mini coloumn transferred to a new centrifuge tube, add 200 uL Buffer AE,
in centrifugation for 1 minute at a speed of 6000 g (8000 rpm).

2. Molecular Detection of Wuchereria bancrofti in mosquito Body (Molecular

Laboratory, 2017).

2.1. PCR tube was prepared, coded according to the isolation of DNA code will
be in-PCR
2.2. Mix Reagent prepared according to the number of samples to be examined
such as the table below:

Reagent Volume per reaction

Go Green 12.5 uL Taq

Primary NV-1 1 uL

Primary NV-2 1 uL

ddH2O 5.5 uL

Total per reaction of 20 uL

2.3. Mix reagents divided per tube PCR 20 uL
2.4. Template DNA ditambahkah 5 uL to each tube
2.5. The negative control was added to thetube negative control and a positive
control in the positive control tube.
2.6. Dospining down until no sample or reagent on the wall of the tube.
2.7 Samples in-PCR on a machine Thermocycle with the following settings:

1. Hot Start
94 ° C 3 min

2. Step Cycle 35 cycles

94 ° C 1 minute
55oC 1 minute

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 72oC 2 minutes

3. Final step
94 ° C 1 minute
55oC 1 minute
72oC 10 mins

4. Hold
12 oC ∞

2.8 Sample results of PCR and then proceed to the process of electrophoresis
and tested positive for the electrophoresis results when the target band is
188 bp
3. Molecular Detection of Wuchereria bancrofti in mosquito Body
(Molecular Laboratory, 2017).

3.1. PCR tube was prepared, coded according to the isolation of DNA code will
be in-PCR
3.2. Mix Reagent prepared according to the number of samples to be examined
such as the table below:

Reagent Volume per reaction

Taq Go Green 12.5 uL

Hha-1 Forward Primer 1 uL

Reverse primer Hha-1 1 uL

ddH2O 5.5 uL

Total per reaction of 20 uL

3.3. Mix reagents divided per tube PCR 20 uL
3.4. Template DNA ditambahkah 5 uL to each tube
3.5. The negative control was added to thetube negative control and a positive
control in the positive control tube.
3.6. Dospining down until no sample or reagent on the wall of the tube.
3.7 Samples in-PCR on a machine Thermocycle with the following settings:

5
 1. Hot Start
94 ° C 5 minutes

2. Step Cycle 35 cycles

94 ° C 1 minute
55oC 1 minute
72oC 1 minute

3. Final step
72oC 10 mins

4. Hold
12 oC ∞

3.8. Sample results of PCR and then proceed to the process of electrophoresis
and tested positive for the electrophoresis results when the target band is
322 bp

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 III. WORK PLAN

Name / NIM : Fikrie Fauzan / B1A015086

Title PKL : Detection of Filariasis Of Vector Mosquito Virus Method
Using PCR (Polymerase Chain Reaction)

locations : Research and Development of Disease Vector and Reservoir

(B2P2VRP) Salatiga.
Time : January 17 - February 6th, 2018
Field Supervisor : Passed Susanti, SKM, MPH.
Supervising street vendorsDrs. Daniel Joko Wahyono, M. BIOMED.

No. Day date activity

1. Wedn January  The introduction of environmental laboratory and

esday 17, 2018 B2P2VRP Salatiga.
 Make a visit to the library in B2P2VRP

2. Thurs January  Lectures on Japanese encephalitis disease.

day 18, 2018  Lectures on the identification and manufacture mounted
mosquitoes.
 Doing A visit to the library in B2P2VRP.
3. Friday January  Make a visit proteomics and microbiology lab.
19, 2018  Demo on pulsed field gel electrophoresis.
4. Mond January  Mosquito sample preparation.
ay 22, 2018  Preparation and PCR treatment.
 Preparation and electrophoresis treatment.
 Lecture about the Hanta virus.
 Hanta Virus detection by Elisa method.
5. Tuesd February  Public lecture on Hanta Virus and Reservoir disease.
ay 23, 2018  Preparation and PCR treatment.
 Preparation and electrophoresis treatment.
6. Wedn January  Electrophoresis treatment and the reading of the results
esday 24, 2018 with ImageLab
 Preparation of Pre-PCR Mix with Super script III.
 Preparation and PCR treatment.
 Public lecture on Schistosomiasis in Indonesia, bird flu in
Indonesia and leptospirosis in the Lab. Molecular.
7. Thurs January  The public lecture Global Health Research in the Field by
day 25, 2018 Guest Lecture hall Jenna Davidson in the lab. integrated.

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8. Friday January  A visit to the Library B2P2VRP
26, 2018
9. Mond January  Preparation of DNA sample incubation network and
ay 29, 2018 mosquitoes
 Test ELISA detection method
10. Tuesd January  DNA extraction mosquito sample in incubation
ay 30, 2018  Manufacture and preparation of PCR mix PCR
 Treatment of PCR for identification Brugia spp. And
Wuchereria bancrofti.
11. Wedn January  Visualization and analysis of the results of electrophoresis
esday 31, 2018  Visiting the library B2P2VRP
12. Thurs February  Lab visits and Reference Collection
day 1st, 2018  Visit the library B2P2VRP

13. Friday February  Visit the library B2P2VRP

2nd,
2018
14. Mond February  Library visits B2P2VRP
ay 5th, 2018
15. Tuesd February  Make a visit DUVER B2P2VRP
ay 6th, 2018

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 REFERENCE LIST

DAS, PK, SP Pani and K. Krishnamoorthy. 2002. Prospects of elimination of lymphatic

filariasis in India. Indian Council Med. Res. 32, pp. 5-6.
Department of Health, RI, 2002. Guidelines for the Eradication of Filariasis. Directorate
General of PPM PL - Directorate P2B2 Subdit Filariasis and Schistosomiasis.
Jakarta
DITJEN PPM and PL. 2005. Epidemiology Filariasis. DG PPM and PL, Jakarta, MOH.
Molecular Laboratory. 2017. Detection of Filariasis work instructions with the
Polymerase Chain Reaction method. Salatiga: B2P2VRP.
Malewa, HI 2008. Based Diagnosis Filariasis smear Blood Bank. Indonesian Journal of
Clinical Pathology and Medical Laboratory, 15 (1), pp. 34-37.
Santoso & Nungki, HS 2015.Spesies Microfilariae in Patients with Chronic Filariasis In
Microscopic and Polymerase Chain Reaction (PCR) in East Tanjung Jabung.
J. Media Research and Development, 25 (4), pp. 249-256.
Schmidt, GD and LS Roberts. 2000. Foundation of Parasitology. 6th ed. The McGraw
Hill Companies, Inc. pp.
Sholichah, Z. 2009. Threat From Mosquitoes Culex sp. Neglected. J. BALABA, 5 (1),
pp. 21-23.
Yahya, Santoso, Milana Salim, and Maya Arisanti. 2014. Detection of Brugia malayi in
Armigeres subalbatus and Culex quinquefasciatus were infected blood
Filariasis Patients with PCR method. Aspirator, 6 (2), pp. 35-42.