Thanks to Ben D. Tall, Yi Chen, and Thomas Hammak from the U.S. Food and Drug Administration (FDA), Center for Food Safety and Applied Nutrition (CFSAN) for critically reviewing the manuscript. The authors also thank Hannah Lee (research internship program, Joint Institute for Food Safety and Applied Nutrition (JIFSAN), University of Maryland) for laboratory assistance and David Weingaertner (FDA, CFSAN) for preparing the figure of the schematic overview shown in the video.

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The use of specified instrumentation is not an endorsement by the U.S. Food and Drug Administration. The authors certify that there is no conflict of interest with any financial organization regarding the material discussed in this article.

野生の昆虫から食品由来の病原体を検出した以前の研究では、正確に食品や食品関連環境13,15内の単一のハエの存在の食品関連リスクを評価するために必要な情報が含まれない場合がありますプロトコルの多種多様を使用していた23,24。ここでは、この標準化されたプロトコルを使用して、それを検出し、 クロノバクター属種を単離することが可能であることを実証した。、S.エンテリカ、及びL.体表面と野生に巻き込まシングルハエの消化管からリステリア 。昆虫が標的食品媒介病原体の数が少ないと他の土着微生物25,26の高い番号を搬送することができるので、このプロトコルは、ターゲット食品媒介病原体の検出の感度を高めるために特定の培養培地中のサンプルの一次（時には二次）の濃縮を必要とする。 PCRベースの検出システムからの結果がDETEために（約30時間以内に得られた。クロノバクター属属の ction。とS.エンテリカ ）と、当初のサンプルを処理した後（） リステリア菌の検出のために48時間。したがって、このプロトコルは、信頼性、ならびに迅速および食品媒介病原体の存在のための単一のハエをスクリーニングするのに十分な感度である。 生菌のPCR陽性結果の確認および単離は、多くの研究室の標準操作手順の一部である。また、疫学のために、PCR陽性試料からの純粋な細菌培養物をさらに確認し、生化学的、免疫学的、または遺伝的方法を使用して、食品媒介病原体の血清型が要求される。 S.を検出する際に偽陽性は観察されなかったもののエンテリカとL.シングル野生捕獲ハエの体の部分からリステリアは 、このプロトコルを使用して、我々はクロノバクター属属のための偽陽性の50％の税率にまで見つけた。このことは、属CronobactためのPCRベースの検出システムERハエによって運ばれる非常に複雑な細菌叢の中に存在する他の腸内細菌と交差反応することができる。このように、PCR陽性検体から属クロノバクター属の純粋なコロニーの単離および精製は、評価した他の病原体よりも選択メッキを必要とする。 このプロトコルは、主にクロノバクター属種の存在について、個々の野生捕獲ハエをスクリーニングするために標準化されている。、S.エンテリカ、及びL.商業的PCRベースの検出システムを使用してリステリア 。しかし、このプロトコルはまた、容易にそのような腸管出血性大腸菌 O157などの他の食品媒介病原体の存在のために、単一のハエのスクリーン本体部分に適合させた：H7（いずれかの大腸菌 O157を使用して：H7 MP標準アッセイキット又はE.コリ O157：H7リアルタイムアッセイキット）と志賀毒素原性E.非公開大腸菌 （STEC）グループ（リアルタイムSTECスイートを使用）、取得感度&gt; 80％（編データ）。また、このプロトコルは、潜在的疾患（ゴキブリ、アリ）のベクターが知られている他の昆虫からの食品媒介病原体を検出するように構成することができるが、この分野におけるさらなる研究が必要である。 食中毒アウトブレイクの調査は非常に動的であり、特定の状況及び12,27を検討されてローカル環境によって異なる場合があり、多段階のプロセスを含む。それらは将来の病気を予防することによって直ちに公衆衛生の保護を提供するため、これらの研究は重要である。さらに、これらの調査は、食品媒介微生物が広がっていることによって、新しい機構を解明し、研究28のための新しい分野につながる重要な問題を提起することができます。捜査技術ならびに標準化された迅速、かつ高感度なプロトコルは、個々の昆虫からの食品媒介病原体を検出するために必要である。この標準化されたプロトコルは、ワット、無菌的にハエのような虫を収集する機会を開くHICHは環境サンプリングプログラムの一環として、食品媒介病原菌をベクトルできます。このことから得ることができる疫学的情報は昆虫による食品媒介病原体の伝達のメカニズムの正確な状況を作成するのに使うであろう（ すなわち、露光時間の長さ：、着陸ハエ対着陸排便、と吐き戻すことによってフライ）。 決して唯一の適切なシステムを意味することで、最終的に、ここで説明する商業PCRベースの検出システムで使用することは実用的であり、PCR増幅および属レベルのアンプリコンの可視化を簡素化にもかかわらず、それはない。濃縮されたサンプルからの溶解物は、あるいは公的に入手可能な種特異的プライマー対を使用して、食品媒介病原体の存在をスクリーニングするために使用することができる。しかし、検出感度は、それらの使用の前に実証されるべきである。

それらは食品又は食品接触面と器具1に病原体を広げることができるので、昆虫、食品関連疾患の伝達に重要な役割を果たしている。昆虫、ハエ、ゴキブリ、アリ食品媒介病原体の拡散を好む展示行動の中で。これらの動作は、減衰の問題との関連を含んで拒否し、糞、endophily（建物に入る）、およびsynanthropy（人間と同居）2。そのようなサルモネラなどの食中毒の病原体、 リステリア菌 、 カンピロバクター 、 大腸菌 O157：H7、および属クロノバクター属 （以前はエンテロバクター·サカザキ ）のメンバーは、昆虫3-5によって送信されることが報告されている。 Synanthropic汚物は、汚染された身体の表面から病原体を転送することによって機械的に普及食中毒菌を飛ぶ。しかし、ハエの消化管における食品媒介病原体の存在は、3回までとすることができる自分の体の表面（ボディ、頭、脚、翼）5で観察されるものよりも大きい。食品媒介病原体は、体表面6,7に比べて時間の大きい長さのためのハエの消化管にとどまることができるといくつかの例では、それらが増殖することができます、ハエの消化管4,8,9に定着。彼らはさらに排便と逆流10,11を通じて食品媒介病原体を広げることができるので、これはハエのベクトルポテンシャルを向上させます。 今日では、より迅速に食中毒の発生を検出することができる改良された監視システムがある。食品媒介アウトブレイク調査を行っている間、公衆衛生当局は感染源（S）または車両であってもよい食品（S）を探してください。研究者らは、食品が汚染されていた方法を見つけるために関係する施設（または施設）の環境アセスメントを実施することができると調査12の一部としてのサンプルを収集することがあります。 Desp科学文献、食品媒介病原体のキャリアとしての昆虫に関する特定の食中毒の発生が挑戦している原因となる病原体のベクトルとして昆虫をリンクするの膨大な量をITE。昆虫は無菌的に食品媒介アウトブレイク調査時の環境サンプリングプログラムの一環として収集されていないので、これは主にです。昆虫、環境サンプリング手順は、単一の昆虫が所定の位置にする必要があるから、食品媒介病原体を検出するために、標準化された迅速、高感度かつ信頼性のあるプロトコルの一部として、食品媒介病原体の拡散を有利挙動を示すこと、特にそれらを含めます。 昆虫からの食品媒介病原体の検出のための従来のメッキ技術は面倒であり、昆虫の生得的な共生微生物の急速な成長を克服するために別の培養培地中の標的細菌の競争力の成長に依存。 BAに昆虫を関連付けた研究のほとんどcterial病原体は、一緒に、いくつかの昆虫をプールではなくごと個別に病原体の存在を同定することにより方法の感度が増加している。したがって、これらの研究は、病原体が13-18発見された昆虫の体の部分を区別しませんでした。これは、疫学的な意味を有していてもよいし、異なる緩和戦略につながる可能性がある食品媒介病原体を体表面に、または個々の昆虫の消化管に位置しているかどうかを同定する能力が重要である。機械的なベクターとしては、逆流および食品に排便ものハエは感染の潜在的に高いレベルで病原体を伝達する確率を増加させるのに対し、短時間食品土地のみ、自分の体の表面から細菌の低レベルを移すことが飛ぶ。したがって、個々の昆虫あたり食品媒介病原体の有病率を推定し、その昆虫、細菌のpの身体部分を区別することが重要である<html> athogenが位置しています。 食品媒介病原体を検出するために培養非依存的方法の使用がますます実施されているにもかかわらず、それらは商業的に単一の昆虫からの食品媒介病原体を検出するために使用されていない。現在、業界や規制当局によって使用されている食品からの食品媒介病原体の迅速な検出のために市販されている検証済みの分子のプロトコルがあります。これらの方法は、食品サンプルの様々な病原体の検出のためのDNAベースのシステムを含む。分子のプロトコルは、従来のメッキ法よりも高速であるが、試料の濃縮は、依然として、ポリメラーゼ連鎖反応（PCR）に必要な細菌性病原体の10 2コロニー形成単位（CFU）に基づく方法19の感度レベルを得るために必要とされる。また、PCR陽性試料からの純粋な細菌コロニーの単離は、適切な方法を使用して病原体を確認するために必要とされる。 コンテンツ &quot;&gt;このプロトコルの目的は、体表面からの食中毒菌の検出および単ハエの消化管のための食品及び環境試料から病原体を検出し、さらにそれらを単離するために使用される市販のPCRベースのシステムを標準化することであるプロトコルのsamples.The感度からの病原体は、ここで説明した第1の実験的に各細菌性病原体の連続希釈を与えた実験室で飼育大人のイエバエ（ イエバエ ）で較正した。標準化されたプロトコルは、その後100野生捕獲を調査するために使用された自分の体の表面および/または消化管からの食品媒介病原体の存在のために飛ぶ。これは標準化されたプロトコルは、公衆衛生研究所の食品媒介を行う際に環境サンプリングプログラムの一部としてそれらを収集する可能性を考慮して、昆虫によってもたらされる健康上の脅威を検出することを可能にするアウトブレイク調査。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Bismuth sulfite (BS) agar</td>
			<td>Fisher Scientific</td>
			<td>R452402</td>
			<td>*Multiple suppliers.</td>
		</tr>
		<tr>
			<td>Brain heart infusion (BHI) broth</td>
			<td>Becton, Dickson and Company</td>
			<td>299070</td>
			<td>*Pre-warmed to 37&#176;C. Multiple suppliers.</td>
		</tr>
		<tr>
			<td>Brilliance Listeria agar (BLA)</td>
			<td>Fisher Scientific</td>
			<td>CM1080B</td>
			<td>*Multiple suppliers.</td>
		</tr>
		<tr>
			<td>Buffered peptone water (BPW)</td>
			<td>Becton, Dickson and Company</td>
			<td>212367</td>
			<td>*Pre-warmed to 37&#176;C or 42&#176;C. Multiple suppliers.</td>
		</tr>
		<tr>
			<td>Brilliance Cronobacter agar (Druggan-Forsythe-Iversen formulation/DFI)</td>
			<td>Fisher Scientific</td>
			<td>CM1055B</td>
			<td>*Multiple suppliers.</td>
		</tr>
		<tr>
			<td>chromID Sakazakii Agar</td>
			<td>bioM&#233;rieux</td>
			<td>43741</td>
			<td>*Call for information: 800.682.2666</td>
		</tr>
		<tr>
			<td>R &#38; F Enterobacter sakazakii (Cronobacter) Chromogenic Plating Medium</td>
			<td>R &#38; F Laboratories</td>
			<td>Various</td>
			<td>*Call for information: +1.630.969.530</td>
		</tr>
		<tr>
			<td>R &#38; F Enterobacter sakazakiii Enrichment Broth and supplement</td>
			<td>R &#38; F Laboratories</td>
			<td>Various</td>
			<td>*Call for information: +1.630.969.530</td>
		</tr>
		<tr>
			<td>Hektoen enteric (HE) agar</td>
			<td>Fisher Scientific</td>
			<td>OXCM0419B</td>
			<td>*Multiple suppliers.</td>
		</tr>
		<tr>
			<td>24 Listeria enrichment broth (24LEB)</td>
			<td>Oxoid</td>
			<td>CM1107</td>
			<td>*Freshly prepared at room-temperature. Multiple suppliers.</td>
		</tr>
		<tr>
			<td>Listeria selective enrichment supplement</td>
			<td>Oxoid</td>
			<td>SR0243</td>
			<td>*Multiple suppliers.</td>
		</tr>
		<tr>
			<td>Novobiocin</td>
			<td>Fisher Scientific</td>
			<td>OXSR0181E</td>
			<td>*Multiple suppliers. Store at 2-8 &#176;C</td>
		</tr>
		<tr>
			<td>Vancomycin hydrochloride hydrate</td>
			<td>Sigma Aldrich</td>
			<td>861987</td>
			<td>Store at 2-8 &#176;C</td>
		</tr>
		<tr>
			<td>Cefsulodin sodium salt hydrate</td>
			<td>Sigma Aldrich</td>
			<td>C8145</td>
			<td>Store at 2-8 &#176;C</td>
		</tr>
		<tr>
			<td>Rappaport-Vassiliadis (RV) medium</td>
			<td>Fisher Scientific</td>
			<td>CM0669B</td>
			<td>*Multiple suppliers.</td>
		</tr>
		<tr>
			<td>Tetrathionate (TT) Broth</td>
			<td>Becton, Dickson and Company</td>
			<td>249120</td>
			<td>*Multiple suppliers.</td>
		</tr>
		<tr>
			<td>Trypticase soy agar (TSA)</td>
			<td>Becton, Dickson and Company</td>
			<td>236930</td>
			<td>*Multiple suppliers.</td>
		</tr>
		<tr>
			<td>Xylose lysine desoxycholate (XLD) agar</td>
			<td>Becton, Dickson and Company</td>
			<td>221284</td>
			<td>*Multiple suppliers.</td>
		</tr>
		<tr>
			<td>API Biochemical identification system</td>
			<td>bioM&#233;rieux</td>
			<td>Various</td>
			<td>*Call for information: +1.800.682.2666</td>
		</tr>
		<tr>
			<td>VITEK 2: Product Safety</td>
			<td>bioM&#233;rieux</td>
			<td>Various</td>
			<td>*Call for information: +1.800.682.2667</td>
		</tr>
		<tr>
			<td>BAX System Q7</td>
			<td>DuPont</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>BAX E. sakazakii Standard assay kit</td>
			<td>DuPont</td>
			<td>D11801836</td>
			<td>*</td>
		</tr>
		<tr>
			<td>BAX L. monocytogenes 24E assay kit</td>
			<td>DuPont</td>
			<td>D13608125</td>
			<td>*</td>
		</tr>
		<tr>
			<td>BAX Salmonella 2 Standard assay kit</td>
			<td>DuPont</td>
			<td>D14368501</td>
			<td>*</td>
		</tr>
		<tr>
			<td>Capping tool</td>
			<td>DuPont</td>
			<td>D11677028</td>
			<td></td>
		</tr>
		<tr>
			<td>Decapping tool</td>
			<td>DuPont</td>
			<td>D11134095</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR tube rack/holder</td>
			<td>DuPont</td>
			<td>D12701663</td>
			<td></td>
		</tr>
		<tr>
			<td>Featherweight forceps, wide tip</td>
			<td>BioQuip</td>
			<td>4750</td>
			<td>Sterilize before use. Multiple suppliers.</td>
		</tr>
		<tr>
			<td>Fine point, straight tip forceps</td>
			<td>BioQuip</td>
			<td>4731</td>
			<td>Sterilize before use. Multiple suppliers.</td>
		</tr>
		<tr>
			<td>Zirconia/silica beads, 0.5 mm</td>
			<td>Bio Spec Products, Inc.</td>
			<td>11079105z</td>
			<td>Multiple suppliers.</td>
		</tr>
		<tr>
			<td>Petri dishes - 60X15mm</td>
			<td>Fisher Scientific</td>
			<td>08-772B</td>
			<td>Multiple suppliers.</td>
		</tr>
		<tr>
			<td>Disposable inoculating loops, 10&#181;L</td>
			<td>Fisher Scientific</td>
			<td>22-363-606</td>
			<td>Multiple suppliers.</td>
		</tr>
		<tr>
			<td>L-shaped cell spreaders</td>
			<td>Fisher Scientific</td>
			<td>14-665-230</td>
			<td>Multiple suppliers.</td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes, 2 ml</td>
			<td>Fisher Scientific</td>
			<td>Various</td>
			<td>Sterilize before use when needed. Secure lid is preferred. Multiple suppliers.</td>
		</tr>
		<tr>
			<td>Cluster tubes</td>
			<td>Fisher Scientific</td>
			<td>05-500-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Cluster tubes caps</td>
			<td>Fisher Scientific</td>
			<td>05-500-23</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hypochlorite (Liquid chlorine bleach)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>*Dilute to 0.05% with water. Multiple suppliers.</td>
		</tr>
		<tr>
			<td>Sterile deionized water</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Multiple suppliers.</td>
		</tr>
		<tr>
			<td>Sterile distilled water</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Multiple suppliers.</td>
		</tr>
		<tr>
			<td>Ethyl alcohol 190 proof</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>*Dilute to 70% with water when needed. Multiple suppliers.</td>
		</tr>
		<tr>
			<td>Genie cell disruptor, 120V - for 1.5ml and 2.0ml microtubes</td>
			<td>Scientific Industries, Inc.</td>
			<td>SI-D238</td>
			<td>Multiple suppliers.</td>
		</tr>
		<tr>
			<td>Heating block</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Multiple suppliers.</td>
		</tr>
		<tr>
			<td>Cooling block</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Multiple suppliers.</td>
		</tr>
		<tr>
			<td>Recirculating water bath</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Multiple suppliers.</td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Multiple suppliers.</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Multiple suppliers.</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Multiple suppliers.</td>
		</tr>
	</tbody>
</table>

detection of foodborne bacterial pathogens from individual filth flies

There is unanimous consensus that insects are important vectors of foodborne pathogens. However, linking insects as vectors of the pathogen causing a particular foodborne illness outbreak has been challenging. This is because insects are not being aseptically collected as part of an environmental sampling program during foodborne outbreak investigations and because there is not a standardized method to detect foodborne bacteria from individual insects. To take a step towards solving this problem, we adapted a protocol from a commercially available PCR-based system that detects foodborne pathogens from food and environmental samples, to detect foodborne pathogens from individual flies.Using this standardized protocol, we surveyed 100 wild-caught flies for the presence of Cronobacter spp., Salmonella enterica, and Listeria monocytogenes and demonstrated that it was possible to detect and further isolate these pathogens from the body surface and the alimentary canal of a single fly. Twenty-two percent of the alimentary canals and 8% of the body surfaces from collected wild flies were positive for at least one of the three foodborne pathogens. The prevalence of Cronobacter spp. on either body part of the flies was statistically higher (19%) than the prevalence of S. enterica (7%) and L.monocytogenes (4%). No false positives were observed when detecting S. enterica and L. monocytogenes using this PCR-based system because pure bacterial cultures were obtained from all PCR-positive results. However, pure Cronobacter colonies were not obtained from about 50% of PCR-positive samples, suggesting that the PCR-based detection system for this pathogen cross-reacts with other Enterobacteriaceae present among the highly complex microbiota carried by wild flies. The standardized protocol presented here will allow laboratories to detect bacterial foodborne pathogens from aseptically collected insects, thereby giving public health officials another line of evidence to find out how the food was contaminated when performing foodborne outbreak investigations.

C.の- （10 8 CFU / mlの10 2）このプロトコルは、最初に実験的に連続希釈を含む液体フライ食品（2％の粉末ミルク）で24時間飼育した実験室で飼育イエバエのセットに校正したサカザキ、S.エンテリカ、L.リステリア 、またはC.ジェジュニ （N = 21、各細菌性病原体のために）。 PCRベースのシステムは、身体の表面および実験供給する単一の消化管からの細菌の最低レベル（10 2 CFU / ml）を検出することになるまで濃縮媒体、ならびにインキュベーションの時間および温度は、それぞれの食品媒介病原体のために調整した飛ぶ。プロトコルセクションに記載の濃縮培地および条件を用いて、PCRベースのシステムはCを検出サカザキ、S.エンテリカ 、及びL.細菌の接種材料&gt; 10 3 CFU / mlである（ 図1A）が供給されるハエの100パーセントの体表面からリステリア 。ハエは、10を与えたとき2 CFU / mlで、Cの検出の割合サカザキ、S.エンテリカ 、及びL.彼らの体表面からのリステリアは、それぞれ100％、66％、33％、（ 図1A）であった。 PCRベースのシステムは、また、割合≥33％（ 図1B）でのすべての細菌の濃度を与えたハエの消化管からこれら3食品媒介病原体を検出した。しかし、Cの検出実験室で飼育ハエが実験最高細菌接種（10 8 CFU / ml）を含む液体食品を与えたときジェジュニにのみ達成された。したがって、C.ジェジュニは 、このPCRベースの検出システムを使用して、個々のハエsynanthropic汚物から試験することができる食品媒介病原体の群から除外した。 この標準化されたプロトコルでは、我々はクロノバクター属属の有病率を決定することができました。、S.エンテリカ、及びL.体表面および/ ​​または目のリステリア個別に無菌的ワシントン首都圏に位置して10都市のレストランの収集のエリアから摘発された100野生のハエのE消化管、DC 5収集された汚物のハエはM.を含む少なくとも6種の代表だったイエバエ （47％）、 ルシリアヒツジキンバエ（33％）、L.セリカータ（14％）、Cochliomyiaのmacellaria（2％）、ニクバエhaemorrhoidalis（2％）、及びOphyraのleucostoma（1％）。 Oneフライのみファミリーレベルに同定された（ハナバエ科; 1％）。表面消毒プロトコルは、細菌の増殖は、各個々のハエの最後の消毒すすぎ水のためのTSAプレート上で観察されなかったので、フライの身体部分との間の交差汚染を回避することに有効であった。このように、区別は、各ハエの体の部分に存在する食品媒介細菌の間で行うことができた。 偽陽性は、体表面のサンプルおよび消化管からは検出されなかった個々のハエS.の検出のためにこの市販のPCRベースのシステムを使用してエンテリカとL.リステリア 、寒天プレート上の生存可能な病原体の確認は、PCR陽性の結果と一致した。しかし、 クロノバクター属種の純粋培養物を単離することは不可能であった。全てのPCR陽性検体から。 PCRベースによるこの病原体のため、検出 （; 9/18 50％）及び消化管（48％; 33分の16）は、単一の野生捕獲ハエのシステムは、体表面からの偽陽性を示した。特定のメディア上にプレートしたランダムに選択されたPCR陰性サンプルは、食品媒介病原体が存在しないことを確認した。 クロノバクター属種を検出するために、この市販のPCRベースのシステムを使用する場合したがって、偽陰性サンプルのいずれからも検出されなかった。、S.エンテリカ 、またはL.リステリア 。 唯一の病原体が単離され、確認されたそれらのPCR陽性検体は陽性とみなしたと統計分析のために含まれています。野生捕獲汚物ハエの消化管における食品媒介病原体の全体的な存在は（DF = 1、P = 0.0087、χ2 = 6.8772）、体表面上よりも有意に高かった。消化管の22％、回収野生ハエの体表面の8％の三食品媒介病原体（ 図2）の少なくとも1つについて陽性であった。全体的に、 クロノバクター属種の有病率。 S.の有病率よりも、体表面または収集されたハエの消化管のいずれかで（フィッシャーの正確確率検定p = 0.0165 19％）、統計的に高かったエンテリカ （7％）とリステリア菌 （4％）。各細菌性病原体（ 図3のためのハエの体の部分の間の一対比較を行うときしかし、統計的な差異は観察されなかった。 クロノバクター属のためのフィッシャーの正確検定p = 0.1464、P = 0.1184およびp = 0.6212属、S.エンテリカ 、及びL.モノサイトゲネス 、それぞれ）。ハエのいずれも、評価され、3つすべての病原体について陽性であったなかった。しかし、ハエ（2 L.のヒツジキンバエと1 のL.セリカータ ）を搭載してサルモネラ属菌の3。とL.表面上または消化管におけるリステリア 。 <img alt="図1" src="/files/ftp_upload/52372/52372fig1.jpg" /> （A）は、本体表面と（B）の異なる細菌接種材料を含有する液体飼料を与えた個々の実験室飼育イエバエの消化管からクロノバクター属のサカザキ 、 サルモネラ菌、リステリア菌、およびカンピロバクター·ジェジュニの図1.検出レベル （N =各細菌性病原体のための21、N =各細菌濃度あたり3）。 <img alt="図2"SRC = &quot;/ファイル/ ftp_upload / 52372 / 52372fig2.jpg&quot; /&gt; ボディ表面および個々のハエの消化管の図2.パーセンテージは、ターゲット食品媒介病原体のいずれかのための肯定的な発見した。 <img alt="図3" src="/files/ftp_upload/52372/52372fig3.jpg" /> クロノバクター属属の図3.有病率。、 サルモネラ菌、体表面からのリステリア菌とsynanthropic野生捕獲ハエの消化管。報告されたp値は、体表面と各細菌病原体のために消化管間の対比較からのものである（フィッシャーの正確確率検定、p値 &lt;0.05は、統計的有意性を示す）。著作権©アメリカ微生物学会、応用のジャーナルと環境微生物学78（22）：7891から902、2012、DOI：10.1128 / AEM.02195-12。

Watch this Scientific Journal Video about Detection of Foodborne Bacterial Pathogens from Individual Filth Flies at JoVE.com

Detection of Foodborne Bacterial Pathogens from Individual Filth Flies

A PCR-based protocol was adapted to detect Cronobacter spp., Salmonella enterica, and Listeria monocytogenes from body surfaces and alimentary canals of individual wild-caught flies. The goal of this protocol is to detect and isolate bacterial pathogens from individual insects collected as part of an environmental sampling program during foodborne outbreak investigations.

個々のフィルスのハエから食中毒細菌性病原体の検出

There is unanimous consensus that insects are important vectors of foodborne pathogens. However, linking insects as vectors of the pathogen causing a ...

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18.2K ビュー.  U.S. Food and Drug Administration. A PCR-based protocol was adapted to detect Cronobacter spp., Salmonella enterica, and Listeria monocytogenes from body surfaces and alimentary canals of individual wild-caught flies. The goal of this protocol is to detect and isolate bacterial pathogens from individual insects collected as part of an environmental sampling program during foodborne outbreak investigations.

ビデオ: Detection of Foodborne Bacterial Pathogens from Individual Filth Flies

Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water

This protocol describes rapid colorimetric detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes (10 L) of agricultural waters. Here, water is filtered through sterile Modified Moore Swabs (MMS), which consist&#160;of a simple gauze filter enclosed in a plastic cartridge, to concentrate bacteria. Following filtration, non-selective or selective enrichments for the target bacteria are performed in the MMS. For colorimetric detection of the target bacteria, the enrichments are then assayed using paper-based analytical devices (&#181;PADs) embedded with bacteria-indicative substrates. Each substrate reacts with target-indicative bacterial enzymes, generating colored products that can be detected visually (qualitative detection) on the &#181;PAD. Alternatively, digital images of the reacted &#181;PADs can be generated with common scanning or photographic devices and analyzed using ImageJ software, allowing for more objective and standardized interpretation of results. Although the biochemical screening procedures are designed to identify the aforementioned bacterial pathogens, in some cases enzymes produced by background microbiota or the degradation of the colorimetric substrates may produce a false positive. Therefore, confirmation using a more discriminatory diagnostic is needed. Nonetheless, this bacterial concentration and detection platform is inexpensive, sensitive (0.1 CFU/ml detection limit), easy to perform, and rapid (concentration, enrichment, and detection are performed within approximately 24&#160;hr), justifying its use as an initial screening method for the microbiological quality of agricultural water.

Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water

A protocol involving integrated concentration, enrichment, and end-point colorimetric detection of foodborne pathogens in large volumes of agricultural water is presented here. Water is filtered through Modified Moore Swabs (MMS), enriched with selective or non-selective media, and detection is performed using paper-based analytical devices (&#181;PAD) imbedded with bacterial-indicative colorimetric substrates.

比色検出の紙ベース<em&gt;エシェリヒア·コリ</em&gt;<em&gt;サルモネラ</em&gt;属、および<em&gt;リステリア菌</em&gt;農業大量の水から

This protocol describes rapid colorimetric detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes (10 L) of ...

環境学

Building Double-decker Traps for Early Detection of Emerald Ash Borer

Emerald ash borer (EAB) (Agrilus planipennis Fairmaire), the most destructive forest insect to have invaded North America, has killed hundreds of millions of forest and landscape ash (Fraxinus spp.) trees. Several artificial trap designs to attract and capture EAB beetles have been developed to detect, delineate, and monitor infestations. Double-decker (DD) traps consist of two corrugated plastic prisms, one green and one purple, attached to a 3 m tall polyvinyl chloride (PVC) pipe supported by a t-post. The green prism at the top of the PVC pipe is baited with cis-3-hexenol, a compound produced by ash foliage. Surfaces of both prisms are coated with sticky insect glue to capture adult EAB beetles. Double-decker traps should be placed near ash trees but in open areas, exposed to sun. Double-decker trap construction and placement are presented here, along with a summary of field experiments demonstrating the efficacy of DD traps in capturing EAB beetles. In a recent study in sites with relatively low EAB densities, double-decker traps captured significantly more EAB than green or purple prism traps or green funnel traps, all of which are designed to be suspended from a branch in the canopy of ash trees. A greater percentage of double decker traps were positive, i.e., captured at least one EAB, than the prism traps or funnel traps that were hung in ash tree canopies.

Effective traps to attract and capture the emerald ash borer (EAB) are a key element of detecting and managing this invasive pest. Double-decker traps, placed in full sun near ash trees, incorporate visual and olfactory cues and were more likely to capture EAB than other trap designs in field trials.

エメラルド灰中くり盤の早期発見のため 2 階建てトラップを構築

Emerald ash borer (EAB) (Agrilus planipennis Fairmaire), the most destructive forest insect to have invaded North America, has killed hundreds of millions ...

Evaluating the Effect of Environmental Chemicals on Honey Bee Development from the Individual to Colony Level

The presence of pesticides in the beekeeping environment is one of the most serious problems that impacts the life of a honey bee. Pesticides can be brought back to the beehive after the bees have foraged on flowers that have been sprayed with pesticides. Pesticide contaminated food can be exchanged between workers which then feed larvae and therefore can potentially affect the development of honey bees. Thus, residual pesticides in the environment can become a chronic damaging factor to honey bee populations and gradually lead to colony collapse. In the presented protocol, honey bee feeding methods are described and applied to either an individual honey bee or to a colony. Here, the insect growth regulator (IGR) pyriproxyfen (PPN), which is widely used to control pest insects and is harmful to the development of honey bee larvae and pupae, is used as the pesticide. The presenting procedure can be applied to other potentially harmful chemicals or honeybee pathogens for further studies.

Herein we present a method to feed pesticide contaminated food to both an individual honey bee and a beehive colony. The procedure evaluates the pesticide effect on individual honey bees by in vivo feeding of basic larval diet and also on the natural condition of beehive colony.

個々のコロニーレベルにミツバチ開発に関する環境化学物質の影響を評価します

The presence of pesticides in the beekeeping environment is one of the most serious problems that impacts the life of a honey bee. Pesticides can be ...

Fluorescent Paper Strips for the Detection of Diesel Adulteration with Smartphone Read-out

Three fluorescent molecular rotors of 4-dimethylamino-4-nitrostilbene (4-DNS) were investigated for their potential use as viscosity probes to indicate the content of kerosene in diesel/kerosene blends, a wide-spread activity to adulterate fuel. In solvents with low viscosity, the dyes rapidly deactivate via a so-called twisted intramolecular charge transfer state, efficiently quenching the fluorescence. Measurements of diesel/kerosene blends revealed a good linear correlation between the decrease in fluorescence and the increase of the fraction of the less viscous kerosene in diesel/kerosene blends. Immobilization of the hydroxy derivative 4-DNS-OH in cellulose paper yielded test strips that preserve the fluorescent indicator&#39;s behavior. Combination of the strips with a reader based on a smartphone and a controlling app allowed to create a simple field test. The method can reliably detect the presence of kerosene in diesel from 7 to 100%, outperforming present standard methods for diesel adulteration.

Here, we present a protocol to detect the adulteration of diesel with kerosene using test strips coated with a fluorescent viscosity probe together with a smartphone-based analysis system.

スマート フォンの読み出しとディーゼルの粗悪品の検出のための蛍光紙のストリップ

Three fluorescent molecular rotors of 4-dimethylamino-4-nitrostilbene (4-DNS) were investigated for their potential use as viscosity probes to indicate ...

Detection of Viruses from Bioaerosols Using Anion Exchange Resin

This protocol demonstrates a customized bioaerosol sampling method for viruses. In this system, anion exchange resin is coupled with liquid impingement-based air sampling devices for efficacious concentration of negatively-charged viruses from bioaerosols. Thus, the resin serves as an additional concentration step in the bioaerosol sampling workflow. Nucleic acid extraction of the viral particles is then performed directly from the anion exchange resin, with the resulting sample suitable for molecular analyses. Further, this protocol describes a custom-built bioaerosol chamber capable of generating virus-laden bioaerosols under a variety of environmental conditions and allowing for continuous monitoring of environmental variables such as temperature, humidity, wind speed, and aerosol mass concentration. The main advantage of using this protocol is increased sensitivity of viral detection, as assessed via direct comparison to an unmodified conventional liquid impinger. Other advantages include the potential to concentrate diverse negatively-charged viruses, the low cost of anion exchange resin (~$0.14 per sample), and ease of use. Disadvantages include the inability of this protocol to assess infectivity of resin-adsorbed viral particles, and potentially the need for the optimization of the liquid sampling buffer used within the impinger.

An anion exchange resin-based method, adapted to liquid impingement-based bioaerosol sampling of viruses is demonstrated. When coupled with downstream molecular detection, the method allows for facile and sensitive detection of viruses from bioaerosols.

陰イオン交換樹脂を用いるバイオエアロゾルからのウイルスの検出

This protocol demonstrates a customized bioaerosol sampling method for viruses. In this system, anion exchange resin is coupled with liquid ...

An Anaerobic Biosensor Assay for the Detection of Mercury and Cadmium

Mercury (Hg) bioavailability to microbes is a key step to toxic Hg biomagnification in food webs. Cadmium (Cd) transformations and bioavailability to bacteria control the amount that will accumulate in staple food crops. The bioavailability of these metals is dependent on their speciation in solution, but more particularly under anoxic conditions where Hg is methylated to toxic monomethylmercury (MeHg) and Cd persists in the rhizosphere. Whole-cell microbial biosensors give a quantifiable signal when a metal enters the cytosol and therefore are useful tools to assess metal bioavailability. Unfortunately, most biosensing efforts have so far been constrained to oxic environments due to the limited ability of existing reporter proteins to function in the absence of oxygen. In this study, we present our effort to develop and optimize a whole-cell biosensor assay capable of functioning anaerobically that can detect metals under anoxic condition in quasi-real time and within hours. We describe how the biosensor can help assess how chemical variables relevant to the environmental cycling of metals affect their bioavailability. The following protocol includes methods to (1) prepare Hg and Cd standards under anoxic conditions, (2) prepare the biosensor in the absence of oxygen, (3) design and execute an experiment to determine how a series of variable affects Hg or Cd bioavailability, and (4) to quantify and interpret biosensor data. We show the linear ranges of the biosensors and provide examples showing the method's ability to distinguish between metal bioavailability and toxicity by utilizing both metal-inducible and constitutive strains.

Here, we present a protocol to use an anaerobic whole-cell microbial biosensor to evaluate how different environmental variables affect the bioavailability of Hg and Cd to bacteria in anoxic environments.

水銀、カドミウムの検出のための嫌気性バイオ アッセイ

Mercury (Hg) bioavailability to microbes is a key step to toxic Hg biomagnification in food webs. Cadmium (Cd) transformations and bioavailability to ...

A Precise and Autonomous System for the Detection of Insect Emergence Patterns

Existing systems to measure insect emergence patterns have limitations; they are only partially automated and are limited in the maximum number of emerging insects they can detect. In order to obtain precise measurement of insect emergence, it is necessary for systems to be semi-automated and able to measure large numbers of emerging insects. We addressed these issues by designing and building a system that is automated and can measure emergence of up to 1200 insects. We modified the existing &#34;falling-ball&#34; system using Arduino microcontrollers to automate data collection and expand the sample size through multiple data channels. Multiple data channels enable the user to not only increase their sample size, but also allows for multiple treatments to be run simultaneously in a single experiment. Furthermore, we created an R script to automatically visualize the data as a bubble plot, while also calculating the median day and time of emergence. The current system was designed using 3D printing so the user can modify the system to be adjusted for different species of insects. The goal of this protocol is to investigate important questions in chronobiology and stress physiology, using this precise and automated system to measure insect emergence patterns.

Measurement of insect emergence patterns requires precision. Existing systems are only semi-automated and sample size is limited. We addressed these issues by designing a system using microcontrollers to precisely measure the time of emergence of large numbers of emerging insects.

昆虫の出現パターンの検出の正確かつ自律システム

Existing systems to measure insect emergence patterns have limitations; they are only partially automated and are limited in the maximum number of ...

Individual Culturing of Tigriopus Copepods and Quantitative Analysis of Their Mate-guarding Behavior

Copepods of the genus Tigriopus, which are common zooplankton in rocky tide pools, show precopulatory mate-guarding behavior where a male clasps a potential mate to form a pair. While this phenomenon has attracted interest of researchers, methods for its analysis have not been well described. Here we describe procedures for: 1) individual culturing and staging of Tigriopus juveniles and adults, and 2) video-based analysis of their mate-guarding behavior. The culturing method enables experimental control of paring experience of animals as well as the ability to track their development before behavioral tests. The analysis method allows quantitative evaluation of several aspects of the mate-guarding behavior, including capturing attempts by males and swimming trajectory of mate-guarding pairs. Although these methods were originally established for ethological studies on Tigriopus, with proper modifications they can also be applied to studies of other zooplankton in different research fields, such as physiology, toxicology, and ecological genetics.

Individual Culturing of Tigriopus Copepods and Quantitative Analysis of Their Mate-guarding Behavior

Mate-guarding behavior plays an important role in reproduction of intertidal copepods of the genus Tigriopus. However, methods for studying this behavior have not been well described. Here we describe methods for: 1) individual culture of virgin Tigriopus animals, and 2) quantitative analysis of their mate-guarding behavior.

Tigriopusカイアシ類と仲間を守る行動の定量分析の個別培養

Copepods of the genus Tigriopus, which are common zooplankton in rocky tide pools, show precopulatory mate-guarding behavior where a male clasps a ...

Detection of Tilapia Lake Virus Using Conventional RT-PCR and SYBR Green RT-qPCR

The aim of this method is to facilitate the fast, sensitive and specific detection of Tilapia Lake Virus (TiLV) in tilapia tissues. This protocol can be used as part of surveillance programs, biosecurity measures and in TiLV basic research laboratories. The gold standard of virus diagnostics typically involves virus isolation followed by complementary techniques such as reverse-transcription polymerase chain reaction (RT-PCR) for further verification. This can be cumbersome, time-consuming and typically requires tissue samples heavily infected with virus. The use of RT-quantitative (q)PCR in the detection of viruses is advantageous because of its quantitative nature, high sensitivity, specificity, scalability and its rapid time to result. Here, the entire method of PCR based approaches for TiLV detection is described, from tilapia organ sectioning, total ribonucleic acid (RNA) extraction using a guanidium thiocyanate-phenol-chloroform solution, RNA quantification, followed by a two-step PCR protocol entailing, complementary deoxyribonucleic acid (cDNA) synthesis and detection of TiLV by either conventional PCR or quantitative identification via qPCR using SYBR green I dye. Conventional PCR requires post-PCR steps and will simply inform about the presence of the virus. The latter approach will allow for absolute quantification of TiLV down to as little as 2 copies and thus is exceptionally useful for TiLV diagnosis in sub-clinical cases. A detailed description of the two PCR approaches, representative results from two laboratories and a thorough discussion of the critical parameters of both have been included to ensure that researchers and diagnosticians find their most suitable and applicable method of TiLV detection.

This protocol diagnoses Tilapia Lake Virus (TiLV) in tilapia tissues using RT-PCR methodologies. The entire method is described from tissue dissection to total RNA extraction, followed by cDNA synthesis and detection of TiLV by either conventional PCR or quantitative PCR using dsDNA binding a fluorescent binding dye.

ティラピア湖ウイルスを使用して従来 RT-PCR および SYBR の検出グリーン Rt-qpcr

The aim of this method is to facilitate the fast, sensitive and specific detection of Tilapia Lake Virus (TiLV) in tilapia tissues. This protocol can be ...

Reproducible dsRNA Microinjection and Oviposition Bioassay in Mosquitoes and House Flies

Synthetic dsRNAs, used to induce RNA interference, may have dose dependent phenotypic effects. These effects are difficult to define if the dsRNAs are delivered using a non-quantitative method. Accurate delivery of known quantities of nucleic acids or other chemicals is critical to measure the efficacy of the compound being tested and to allow reliable comparison between compounds.
Here we provide a reproducible, quantitative microinjection protocol that ensures accurate delivery of specific doses of dsRNA, reducing the mortality typically induced by injection injury. These modifications include the addition of Rhodamine B, a graduated injection needle, and an improved recovery method borrowed from Isoe and Collins. This method allows calculation of dose responses and facilitates comparisons between compounds. Versions of this method have been successfully used on three genera of mosquitoes as well as house flies to assess the reduction in fecundity resulting from gene silencing of ribosomal RNA transcripts.
This protocol provides strategies to reduce several challenges of small insect microinjection. Together, mechanical delivery of dsRNAs accompanied by visual verification, identification of effective locations for delivery, and inclusion of a post-injection recovery period ensure accurate dosing and low injury mortality. This protocol also describes an oviposition bioassay for uniform determination of effects on fecundity.

This protocol describes a microinjection methodology that we have standardized and used for several years to deliver specific quantities of nucleic acids directly to the hemolymph of mosquitoes and house flies. This protocol results in minimal injection mortality and allows dose correlated measurements of fecundity.

再現可能な dsRNA のインジェクションと蚊とハエの産卵生物検定

Synthetic dsRNAs, used to induce RNA interference, may have dose dependent phenotypic effects. These effects are difficult to define if the dsRNAs are ...