Multiplex Diagnosis kit for Zika, Dengue, …...Zika virus (ZIKV) - 30-fold increase in global incidence over the past 50 years. - 75% of the global population exposed to dengue are

Multiplex Diagnosis kit for Zika, Dengue,

Chikungunya and Japanese Encephalitis Viruses

using

Printed Array Strip based on

Single-stranded Tag Hybridization method

〇 Takahiro Haruyama, Michiko Kumakura, Chun Ren Lim,

Mitsuo Kawase and Nobuyuki Kobayashi

May 3, 2017, Annecy, France

Mosquito-borne virus

● Dengue virus (DENV)

● Chikungunya virus (CHIKV)

● Japanese encephalitis virus (JEV)

● Zika virus (ZIKV)

- 30-fold increase in global incidence over the past 50 years.

- 75% of the global population exposed to dengue are in the Asia-Pacific region.

- Outbreaks of Zika virus disease have been recorded in Africa, the Americas,

Asia and the Pacific. Since 2015, 62 countries and territories reported mosquito

transmitted Zika virus.

- WHO announced a Public Health Emergency of International Concern (PHEIC)

on 1 February 2016.

- ZIKV infection related to Microcephaly.

- Sexual transmission.

etc.

Affected Region

CHIKV

JEV ZIKV

DENV

All of these maps are represented in WHO’s web site

Local outbreaks of dengue fever in Japan

Apr 29, 2015 / the japan times news

In the summer of 2014, outbreaks of dengue

fever occurred in Tokyo’s Yoyogi Park and

became the nation’s first for 70 years.

・BSL2 / BSL3

・Skills & Materials

related to virus

・STH-PAS technology

・R&D of Diagnostic systems

Mosquito-borne virus detection (MBVD) kits

Research & Development

Nagasaki University Tohoku University

http://avss.jp/

http://www.t-bioarray.com/

http://www.dna-chip.co.jp/

Our Objective

2. Initial clinical symptoms of Dengue, Zika, Chikungunya and Japanese Encephalitis

virus infections are similar.

⇒ be able to distinguish these viruses

⇒ be able to detect at early stage of infection by highly

sensitive than ELISA or Immunochomato-test (IC)

We need more effective diagnosis system to…

1. These mosquito-borne virus diseases are most common in developing countries.

⇒ The cost should be low

⇒ Simple and easy detection

Core Technology “STH-PAS”

STH (Single-strand Tag Hybridization)-PAS (Printed Array-Strip)

Tag No. Target

① DENV (type 1~4)

② CHIKV

③ JEV

④ ZIKV

e.g.

AMR(Bacterial Disease):http://www.amr-inc.jp/

Disease Cocktail name Pathogen

Respiratory Cocktail

Bacterial pharingo-

bronchitis

Mycoplasma pneumoniae DnaJ

Corynebacterium diphtheriaeToxin

Legionella pneumophila DnaJ

Chlamydophila pneumoniae DnaJ

Child tonsilitis

Staphylococcus aureus FtsY

Staphylococcus aureus MRSA

Staphylococcus aureus BlaZ

Streptococcus pyogenes DnaJ

Child bronchitis Bordetella pertussis pertactin1

MycobacteriumMycobacterium avium group

Mycobacterium tuberculosis complex

Tuberculosis Mycobacterium tuberculosis complex

MAC compleX Mycobacterium avium complex

kansasi -gastri Mycobacterium kansasii-gastri

Abscessus Mycobacterium abscessus

Pharingitis/broncho.pneu

monia

Mycoplasma pnemoniae

Chlamydophila pneumoniae

Legionella pneumophila

Alveolar pyralia

Porphylomonas gingivalis

Treponema denticola

Tannerella forsythus

Bioterrolism-Pneumonia

Yersinia pestis_pseudotuberculosis

Francisella tularensis

Bacillus anthracis PA

Burkholderia pseudomallei


Digestive

tract

Cocktail

Diarrheal stool

Shigella/Salmonella

Escherichia coli EHEC1-S.dysenteriae 01

Escherichia coli EHEC2

Escherichia coli EIEC-Shigella spp.

Salmonella enterica InvA

Diarrheal stool

(Campylobacter）

Campylobacter jejuni

Campylobacter coli

Campylobacter fetus

Vibrio

parahaemolyticus

Vibrio parahaemolyticus TDH

Vibrio parahaemolyticus dnaJ

Vibrio choleraVibrio cholerae-CT

Vibrio cholerae dnaJ

Waterly DiarrheaEscherichia coli ETEC ST 1a human

Escherichia coli ETEC LT

Vibrio cholerae-CT3

Protozoal DiarrheaEntoamoeba histolyticum

Cryptosporidium parvum Hsp70-2

Giardia lambria HSP

Enterotoigenic

Escherichia coli -B

Escherichia coli ETEC LT

Escherichia coli ETEC ST 1a human

Escherichia coli ETEC ST 1b porcin

Escherichia coli ETEC ST2 porcin

Yersinial enterocolitisYersinia enterocolitica dnaJ

Yersina pestis-pesudotuberculosis dnaJ

Yersinia pestisYersina pestis Fl antigen

Yersina pestis Pla

Typhoid fever Salmonella enterica var Typhi Vi viaB

Salmonella enterica var Typhi InvA

Staphylococcus aureus

enterotoxin groupA

Enterotoxin A

Enterotoxin B

Enterotoxin C

Enterotoxin D

Staphylococcus

aureus enterotoxin

group B

Enterotoxin E

Enterotoxin G

Enterotoxin H

Enterotoxin I

Bacillus cereus groupB.cereus dnaJ

B.cereus CeuA

Botulinum ABF

Proteolytic

Clostridium botulinum ABF proteolytic

Clostridium botulinum toxin A

Clostridium botulinum toxin B

Clostridium botulinum toxin F

Botulinum-B group;

BEF nonproteolytic

Clostridium botulinum BEF nonproteolytic

Clostridium botulinum toxin B

Clostridium botulinum toxin E

Clostridium botulinum toxin F

Clostridium difficile

Toxin

Clostridium difficile Toxin A

Clostridium difficile Toxin B

Line-ups of Products

Line-ups of Our Products “Single target”Line-ups of Products


STD Cocktail

Urethritis-A

Mycoplasma genitarium DnaJ

Chlamydia trachomatis 16S rRNA

Neisseria gonorrhoae DnaJ

Ureaplasma urealyticum DnaJ

Urethritis-B

Treponema pallidum DnaJ

Haemophilus ducreyi

Trichomonas vaginalis

Vaginal Carrier A

Streptococcus agalactiae

Neisseria gonorrhoae DnaJ

Mycoplasma hominis DnaJ

Trichomonas vaginalis ITS

Vaginal Carrier B

Treponema pallidom 47K

Chlamydia trachomatis

Ureaplasma urealyticum

Mycoplasma genitarium

Vaginal Ulcer

Hemophilus ducreyi

Klebsiella granulomatosis

Herpes simplex !&2

Treponema pallidom

Systemic Infection

Cocktail

Typhoid fever Salmonella Typhi Vi viaB

Salmonella Typhi

TuberculosisMycobacterium avium group

Mycobacterium tuberculosis complex

ListeriaL.monocytogenes group dnaJ

L.monocytogenes hemolysin

LeptospiraLeptospira DnaJ

Leptospira IS1500

BorreliaBorrelia recurrentis

Borrelia burgdorferii

Chlamydia

Chlamydia psittaci holA

Chlamydophila pneumoniae dnaJ

Chlamydophila trachomatis 16SrDNA

EnterococcusVa

Enterococcus VanA

Enterococcus VanB

Enterococcus VanC1

Staphylococcus aureus-Drug

registance

Staphylococcus aureus DnaJ

Staphylococcus aureus MRSA

Staphylococcus aureus BlaZ

A/Bacterial UFO A

Salmonella

Brucella spp.

Campylobacter fetus

B/Bacterial UFO B

Rickettsia

Borrelia burkdorferii

Coxilella burnetii

Orientia tsutsugamushi

Mallei-PseudomalleiBurkholderia mallei-pseudomallei

Burkholderia mallei

Pet-Animal Cocktail

Brucella sppOMP

DnaJ

Mycoplasma agalactiae M.agalactiae DnaJ

Clostridium chauvoei cctA

AVSS(Viral Disease):http://avss.jp/

Category Cocktail name Pathogen

Blood

Dengue Virus

Universal

Dengue Virus

(Subtype 1~4)

Dengue Virus

Subtype at once

Dengue Virus

(Subtype 1~4)

Mosquito-borne

Virus 3

Dengue Virus

(Subtype 1~4)

Zika Virus

Chikungunia Virus

Mosquito-borne

Virus ４

Dengue Virus

(Subtype 1~4)

Zika Virus

Chikungunia Virus

Japanese Encephalitis

Virus

Zika Zika Virus

CHIK Chikungunia Virus

JEVJapanese Encephalitis

Virus

Kit’s component

Outer Bag

Handbook

(kit’s instructions)

STH-PAS Strip

Primers

Developing sol.

w/ Latex beads

DENV-NAC (Nucleic Acid Chromatography)

Universal Primer Kit

・Plasma

・Serum

RNA

extractionRT-PCR

1. Add developer + latex beads 2. Insert strip

After the reaction…

3. Just wait

for 10 min.

Simple & Easy Procedure / No need other equipment / Quick detection

Protocol

Results of Single target kits

DENV-NAC

(Universal)

- + - + - + - +

CHIKV-NAC JEV-NAC ZIKV-NAC

Virus serotype LOD (Limit of Detection)

DENV-1 0.034 copy/assay




Sensitivity of “DENV-NAC Universal Primer Kit”

1 2 43NC

NC: negative control (no template)

DENV subtype

DENV-NAC(Universal)

Kit

DENGUEDAY1 TEST（Mitra）

Dengue NS1 Detect Rapid Test（InBios）

Dengue NS1 Antigen Rapid test（NTBIO）

Dengue2 (IgG/IgM + NS1 Combo) Rapid test（NTBIO）

Limit of Detection

1.7copy/ml

1.4 x 106

copy/ml

1.4 x 106

copy/ml

1.4 x 106

copy/ml

1.4 x 107

copy/ml

“DENV-NAC (Universal) Kit” vs Immunochromato-kits

Line-ups of Our Products “Multiple target”

2. Mosquito-Borne Viruses (MBV) 3 -NAC Kit

3. MBV 4 –NAC Kit

1. Dengue Virus-NAC Subtypes At Once Kit

- Dengue, Chikungunya and Zika viruses At Once -

- Dengue, Chikungunya, Zika

and Japanese encephalitis viruses At Once -

✔ Now on sale (1 & 2 ) ~ Research purpose only.

✔ Price ~ $5 / test

✔ Format~ 20 tests/kit or 100 tests/kit

Under Development, Coming Soon!

- Distinguish subtype 1 ~ 4 At Once -

Results

1. Dengue Virus-NAC Subtypes At Once Kit

1 2 43 NC

DENV subtype

Sub-

typeLOD

(copies/assay)

1 8

2 21

3 8

4 2

Results

2. Mosquito-Borne Viruses (MBV) 3 -NAC Kit

-Dengue, Chikungunya, and Zika viruses At Once-

1 2 3NC

1: DENV

2: CHIKV

3: ZIKV

Advantages/disadvantages of our products

● Advantage

● Disadvantage

- Multiplex detection at once (max. 12 kinds)

- High sensitivity

- Quick & easy detection (10~15 min. after reaction)

- Low cost (~ $ 5.00/test)

- Flexibility against new and emerging viruses (just design

suitable primer sets)

- Target is viral RNA (extraction step is needed)

- Requirement of thermalcycler for RT-PCR

W:210mm×D:170mm×H:170mm

1.8 kg

“Quick Mobile”

QM-0104AStore the RT-PCR

condition in a card

⇒ POCT

Challenges-1

・Plasma

・Serum

RNA

extractionRT-PCR STH-PAS

0

5000

10000

15000

20000

25000

30000

35000

40000

QIAGEN KURABO KANEKA CICA TOYOBO

Co

py n

um

be

r/µ

l

Commercial RNA Extraction Kits

Medium

Plasma

Further Selection &

Optimization of RNA

extraction kits are

needed ！！！！Low recovery

Columntype

Liquidtype

Mag-beadstype

Challenges-2

● Validation

- On going: evaluation and accumulation of the data using clinical samples.

It was collaborated with some institutes in South-East Asia countries.

● Distribution

Real-Time PCR

STH-PAS Strips

+ - Total

+ 91 0 91

- 2 18 20

Total 93 18 111

Parameters Estimate

Sensitivity 97.9%Specificity 100%PPV 100%NPV 90%

- Approval as clinical diagnosis kits

- Know-how for the distribution mechanism and funding to disseminate in

affected nations

Poster presentations at BioJapan 2016@ Yokohama

Thank you!

A Juggler of Viruses

http://avss.jp/

http://www.t-bioarray.com/

Documents

Multiplex Diagnosis kit for Zika, Dengue, …...Zika virus (ZIKV) - 30-fold increase in global incidence over the past 50 years. - 75% of the global population exposed to dengue are