STH-PAS による特定遺伝子の検出(Single strand Tag Hybridization –Printed Strip Array)

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アガロースゲル電気泳動/EtBr 検出感度
Results of electrophoresis
Sample: Bovine DNA(50ng) was used for amplification using PAS①②③④⑤⑥⑦⑧⑨⑩⑪ ⑫ ⑬ ⑭ ⑮primer
set

100bp DNA Ladder

3.5ml apply

Signal intensity of ③was judged to be equivalent to the signal intensity of 300bp band of 100bp ladder markers (14ng).

DNA amounts in lanes⑥：1.75ng⑪：0.0515625ng①：56ng⑦：0.825ng⑫：0.02578125ng②：28ng⑧：0.4125ng ⑬：0.012890625ng③：14ng⑨：0.20625ng ⑭：0.0064453125ng④：7ng⑩：0.103125ng ⑮：0.00322265625ng⑤：3.5ng

Detection limit is 3.5ng DNA (lane ⑤)

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STH-PAS 検出感度
Results：①ver. 1 PAS
Experiments
・PCR products, which were used in the test ofelectrophresis/EtBr detection sensitivity, were usedfor the test of PAS detection sensitivity.

Amounts of DNA applied①：56ng ⑥：1.75ng ⑪：0.052ng②：28ng ⑦：0.83ng ⑫：0.026ng③：14ng ⑧：0.41ng ⑬：0.013ng④：7ng⑨：0.21ng ⑭：0.0064ng⑤：3.5ng ⑩：0.11ng ⑮：0.0032ng②ver.2 Primer trap-PAS
Amounts of DNA applied①：56ng ⑥：1.75ng ⑪：0.052ng②：28ng ⑦：0.83ng ⑫：0.026ng③：14ng ⑧：0.41ng ⑬：0.013ng④：7ng ⑨：0.21ng ⑭：0.0064ng⑤：3.5ng ⑩：0.11ng ⑮：0.0032ng

Detection limit of PAS： ver1 and ver2
showed that the limit is 0.052ng ⑪

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STH-PAS 検出の流れ
Biological sample

Genuine detection?
Wait for 5 min

Extract DNA/RNAtarget 2
Target 1

PCRproducts

PCR using specific primers of different target to detect
wide range of strains in a single PCR
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Positive control

Norovirus, sometimes known as the winter vomiting bug[1] in the UK, is themost common cause of viral gastroenteritis in humans. It affects people of allages.[2] The virus is transmitted by fecally contaminated food or water, by personto-person contact,[3] and via aerosolization of the virus and subsequentcontamination of surfaces.[4] The virus affects around 267 million people andcauses over 200,000 deaths each year; these deaths are usually in less developed
countries and in the very young, elderly and immunosuppressed.[5]Noroviruses (NoV) are a genetically diverse group of single-stranded RNA, nonenveloped viruses belonging to the Caliciviridae family.[25] According to theInternational Committee on Taxonomy of Viruses, the genus Norovirus has one
species, which is called Norwalk virus.[26] Noroviruses can genetically be classified
into five different genogroups (GI, GII, GIII, GIV, and GV), which can be further
divided into different genetic clusters or genotypes. For example, genogroup II, themost prevalent human genogroup, presently contains 19 genotypes. Genogroups I,II and IV infect humans, whereas genogroup III infects bovine species, andgenogroup V has recently been isolated in mice.[28] g
In the present project, the 36 complete sequences of human norovirus (all of thesequences registered at NCBI at 2015/02/22 14:01) were collected and used for
determination of detection primer.
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Examination with host materials

1 2 3 4
1. Human RNA +GII.4 +GII.17
2. Human RNA
3.Human RNA+ GII.4
4. Human RNA+ GII.17

Host RNAGII.4GII.17

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Detection with lower numbers of norovirus RNA moleclues
1 2 3 4
Flow cont’l

PCR: 33 cycles

Signal

1: 100 molecules/RT-reaction
2: 10 molecules/RT-reaction
3: water in RT/ reaction
4: water in PCR (noRT)

is supposed to be a threshold forregular detection
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Zika virus outbreak (2015–present)
As of early 2016, a widespread outbreak of Zika fever, caused by the Zika virus, isongoing, primarily in the Americas. The outbreak began in April 2015 in Brazil, and has
spread to other countries in South America, Central America, Mexico, andtheCaribbean. In January 2016, the World Health Organization (WHO) said the virus
was likely to spread throughout most of the Americas by the end of the year; [2] and inFebruary 2016, the WHO declared the cluster of microcephaly and Guillain–Barrésyndrome (GBS) cases reported in Brazil – strongly suspected to be associated withthe Zika virus outbreak – a Public Health Emergency of International Concern.[3][4][5][6]The Zika virus was first linked with newborn microcephaly during the Brazil Zika virus
outbreak. In 2015, there were 2,782 cases of microcephaly compared with 147 in2014 and 167 in 2013.[68]Zika is a mosquito-borne disease and possibly a sexually transmitted infection.[8] The resurgence of Aedes aegypti'sworldwide distribution over the past 2–3 decades makes it one of the most widely distributed mosquito species.[63] In
2015, Aedes albopictus was present in tropical, subtropical, and temperate regions of the Americas, reaching as far
north as the Great Lakes of North America and, internationally, living alongside Aedes aegypti in some tropical andsubtropical regions.[7]つくば遺伝子研究所

Twelve Zika virus complete genome sequences were found in NCBI nucleotide
database at point of 10th February, 2016. Therefore, all the virus sequences werecollected and primer set was designed to detect all virus sequences registered at thistime point.

Procedures
1. RT using Zika specific RT-primer and mammalian rRNA specific RT-primer
2. PCR using respective species specific primer pairs
3. Electrophoresis using a 2.5% agarose gel.

1,7:100bp marker
2: H2O at RT
3: H2o at PCR
4: Human RNA
5:Zika RNA(portion)
6:Zika RNA(portion)plus HumanRNA

1 2 3 4 5 6 7

2 3 4 5 6PAS解析

Conclusion : Zika viruses are able toつくば遺伝子研究所
be detectedDengue virus
Dengue fever (UK /ˈdɛŋɡeɪ/ or US /ˈdɛŋɡiː/), also known as breakbone fever, is amosquito-borne tropical disease caused by the dengue virus consisting of single positivestrand RNA.

The dengue virus genome (genetic material) contains about 11,000 nucleotide bases,
which code for the three different types of protein molecules (C, prM and E) that form
the virus particle and seven other types of protein molecules (NS1, NS2a, NS2b, NS3,NS4a, NS4b, NS5) that are only found in infected host cells and are required forreplication of the virus.[15][17] There are five[1] strains of the virus, called serotypes, ofwhich the first four are referred to as DENV-1, DENV-2, DENV-3 and DENV-4.[4] The
fifth type was announced in 2013.[1] http://en.wikipedia.org/wiki/Dengue_fever
Currently, we are focusing on the detection of DENV-1, DENV-2, DENV-3 and DENV4

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Collection of dengue virus complete genome sequenceType 1: 49 genome sequences
Type2: 50 genome sequences
Type3: 50 genome sequences

Type4: 50 genome sequences

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PAS system test with host materials 21
Flow control

2345 6
3: Type 1: 49 genome sequences
4: Type2: 50 genome sequences

Host RNA

5: Type3: 50 genome sequences
5: Type4: 50 genome sequences

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Malaria is a mosquito-borne infectious disease of humans and other animals caused byparasitic protozoans (a group of single-celled microorganism) belonging to thegenus Plasmodium.Malaria causes symptoms that typically include fever, fatigue, vomiting,and headaches. In severe cases it can cause yellow skin, seizures, coma or death.
Five species of Plasmodium can infect and be spread by humans.[2] Most deaths arecaused by P. falciparum because P. vivax, P. ovale, and P. malariae generally cause amilder form of malaria.[1][2] The species P. knowlesi rarely causes disease in humans.[1]From wikipedia
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Detection of malaria 5 species rRNA with Mix primer (multiplex RT and PCR)
STH-PAS detection using 1ml of PCR product
DW F K M OM1V

2M, molecular weight marker
1, Human RNA human rRNA RT and PCR primer
2, Human RNA Maralia RT and PCR primers

熱帯熱マラリア原虫（P. falciparum）、
三日熱マラリア原虫（P. vivax）、
四日熱マラリア原虫（P. malariae）、Malaria signal was not detected in RNA from
卵形マラリア原虫（P. ovale）
non-infected human cells (see lane2)
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サルマラリア原虫（P. knowlesi）
For detailed examination, see next pageEbola virus disease
Ebola virus disease (EVD; also Ebola hemorrhagic fever, or EHF), or simply Ebola, is adisease of humans and other primates caused by ebolaviruses. Signs and symptoms
typically start between two days and three weeks after contracting the virus with afever, sore throat, muscular pain, and headaches. Then, vomiting, diarrhea and rashusually follow, along with decreased function of the liver and kidneys. At this time somepeople begin to bleed both internally and externally.[1] The disease has a high risk ofdeath, killing between 25 and 90 percent of those infected, with an average of about 50
percent.[1]Ebolaviruses contain single-stranded, non-infectious RNA genomes.[29]Ebolavirus genomes contain seven genes including 3‘-UTR-NP-VP35-VP40-GP-VP30VP24-L-5’-UTR.[21][30] The genomes of the five different ebolaviruses (BDBV, EBOV, RESTV,SUDV and TAFV) differ in sequence
The natural reservoir for Ebola has yet to be confirmed; however, bats are
considered to be the most likely candidate species.[43]http://en.wikipedia.org/wiki/Ebola_virus_disease

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Collected viral genome sequences
Sudan ebolavirus
http://www.ncbi.nlm.nih.gov/nuccore/55770807
LOCUS
NC_006432
18875 bp cRNA linear VRL 12-NOV-2014
All (10 seqs) the complete genome sequences registered on 2014.12.31
Reston ebolavirus
http://www.ncbi.nlm.nih.gov/nuccore/22789222
LOCUS
NC_004161
18891 bp cRNA linear VRL 12-NOV-2014
All (8 seqs) the complete genome sequences registered on 2014.12.31
Zaire ebolavirus
http://www.ncbi.nlm.nih.gov/nuccore/10313991
LOCUS
NC_002549
18959 bp cRNA linear VRL 12-NOV-2014
11 complete genome sequences randomly-selected from the sequences registered on 2014.12.31
Bundibugyo virus
http://www.ncbi.nlm.nih.gov/nuccore/NC_014373
LOCUS
NC_014373
18940 bp cRNA linear VRL 12-NOV-2014
All (6 seqs) the complete genome sequences registered on 2014.12.31
Tai Forest ebolavirus
http://www.ncbi.nlm.nih.gov/nuccore/302315369
LOCUS
NC_014372
18935 bp cRNA linear VRL 12-NOV-2014
All (2 seqs) the complete genome sequences registered on 2014.12.31
Lloviu cuevavirus
http://www.ncbi.nlm.nih.gov/nuccore/355469071
LOCUS
NC_016144
18927 bp cRNA linear VRL 12-NOV-2014
All (2 seqs) the complete genome sequences registered on 2014.12.31
Marburg marburgvirus
http://www.ncbi.nlm.nih.gov/nuccore/158539108
LOCUS
NC_001608
19111 bp cRNA linear VRL 12-NOV-2014
10 complete genome sequences randomly-selected fromthe sequences registered on 2014.12.31
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Examination of PAS system for ebola virus RNA (Ebola5+2) under the pre-existing
host RNA
Trial 3 (final condition)
Condition: RT65C 60min, 70C 15min
PCR 95C 9min(1cycle), 95C 30sec, 68C 30sec, 72C 30sec (28cycles), 72C 5min( 1cycle)

M 1 2 3 4 5 6 7 8 9 M

1 2 3 4 5 6 7 8 9Virus signals

1.
2.
3.
4.
5.
6.
Host RNA 7.
8.
9.

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Virus RNA mix
Host RNA only (Human)
Bundibugyo virus + hostRNA
Lloviu cuevavirus + hostRNA
Marburg marburgvirus + hostRNA
Reston ebolavirus + hostRNA
Sudan ebolavirus + hostRNATai Forest ebolavirus + hostRNA
Zaire ebolavirus + hostRNAMiddle East respiratory syndrome coronavirus
From Wikipedia, the free encyclopedia(Redirected from MERS-CoV)This article is about the virus. For the disease, see Middle East respiratory syndrome. For
the outbreak, see 2012 Middle East respiratory syndrome coronavirus outbreak.
MERS-CoVMERS-CoV particles as seen by negative stain electron microscopy. Virions containcharacteristic club-like projections emanating from the viral membrane.
Virus classification
Group: Group IV ((+)ssRNA)
Order: Nidovirales
Family: Coronaviridae
Subfamily:Coronavirinae
Genus: Betacoronavirus
Species: MERS-CoVAs of 16 April 2014, MERS-CoV cases have been reported in several countries, including
Saudi Arabia, Malaysia, Jordan, Qatar, the United Arab Emirates, Tunisia, and thePhilippines. The MERS count is 238, with 92 deaths[2] (see 2012 Middle East respiratory
syndrome coronavirus outbreak for a full list of countries and casualities).
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PAS system test with host materials
RT:65CSars2N: Mers RNA* 50ng
Human: humal RNA 50ng
* Partial sequence of MERS –CoV RNA

M 1 2 3M 1 2 3123
Flow control

Host RNAOver exposure

MERS-CoVElectrophoresis
Based on the electrophoresis, thesystem works fine.

Detection method finalized
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Detection of HEV RNA with Printed Strip Array(PAS)
Nested PCR for Genotype 1, G2, G3, G4

１２

Reverse transcription
1:control (no virus RNA)
2: HEV RNA +PCR
1hrPAS detection
5-6minSignal of HEV RNA

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