EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a ...

Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and ...

U.S. Environmental Protection Agency

&#x9690;&#x5B62;&#x5B50;&#x866B;&#x662F;&#x91CD;&#x8981;&#x7684;&#x6C34;&#x6027;&#x539F;&#x751F;&#x52A8;&#x7269;&#x5BC4;&#x751F;&#x866B;&#x53EF;&#x5BFC;&#x81F4;&#x4E25;&#x91CD;&#x8179;&#x6CFB;&#x548C;&#x6B7B;&#x4EA1;&#x4E2D;&#x514D;&#x75AB;&#x529B;&#x4F4E;&#x4E0B;&#x3002;&#x76EE;&#x524D;&#x7684;&#x65B9;&#x6CD5;&#x7528;&#x6765;&#x76D1;&#x89C6;&#x7684;&#x6C34;&#x4E2D;&#x9690;&#x5B62;&#x5B50;&#x866B;&#x5375;&#x56CA;&#x662F; 1622&#x5E74;&#x548C; 1623&#x5E74;&#x57FA;&#x4E8E;&#x663E;&#x5FAE;&#x955C;&#x7684;&#x7F8E;&#x56FD;&#x73AF;&#x4FDD;&#x5C40;&#x65B9;&#x6CD5;&#x3002;&#x8FD9;&#x4E9B;&#x65B9;&#x6CD5;&#x8BC4;&#x4F30;&#x6E90;&#x6C34;&#x57DF;&#xFF0C;&#x866B;&#x5375;&#x56CA;&#x7684;&#x603B;&#x6C34;&#x5E73;&#xFF0C;&#x4F46;&#x5E76;&#x6CA1;&#x6709;&#x786E;&#x5B9A;&#x5375;&#x56CA;&#x6D3B;&#x529B;&#x6216;&#x57FA;&#x56E0;&#x578B;&#x3002;&#x6700;&#x8FD1;&#xFF0C;&#x4E19;&#x952D; monoazide (PMA) &#x5DF2;&#x88AB;&#x7528;&#x4E8E;&#x4E0E;&#x5206;&#x5B50;&#x8BCA;&#x65AD;&#x5DE5;&#x5177;&#x4E00;&#x8D77;&#x786E;&#x5B9A;&#x7269;&#x79CD;&#x548C;&#x8BC4;&#x4F30;&#x53EF;&#x884C;&#x6027;&#x7684;&#x7EC6;&#x83CC;&#x3002;&#x8FD9;&#x9879;&#x7814;&#x7A76;&#x7684;&#x76EE;&#x6807;&#x662F;&#x53D1;&#x5C55;&#x7684;&#x9690;&#x5B62;&#x5B50;&#x866B; PMA-&#xFF08;CryptoPMA-PCR&#xFF09; pcr&#xFF0C;&#x5305;&#x62EC; PMA &#x6CBB;&#x7597; PCR &#x5206;&#x6790;&#x524D;&#x7684;&#xFF0C;&#x4EE5;&#x9632;&#x6B62;&#x4ECE;&#x6B7B;&#x866B;&#x5375;&#x7684; DNA &#x6269;&#x589E;&#x3002;&#x7ED3;&#x679C;&#x8868;&#x660E; PMA &#x7A7F;&#x900F;&#x5176; DNA &#x53EA;&#x6B7B;&#x866B;&#x5375;&#x548C;&#x5757;&#x6269;&#x589E;&#x3002;CryptoPMA pcr &#x8FD8;&#x5177;&#x4F53;&#x53EF;&#x4EE5;&#x68C0;&#x6D4B;&#x6D3B;&#x866B;&#x5375;&#x56CA;&#x5185;&#x7684;&#x6D3B;&#x7684;&#x548C;&#x6B7B;&#x7684;&#x866B;&#x5375;&#x7684;&#x6DF7;&#x5408;&#x4EBA;&#x53E3;&#x3002;&#x66F4;&#x91CD;&#x8981;&#x7684;&#x662F;&#xFF0C;&#x6D3B;&#x866B;&#x5375;&#x56CA;&#xFF0C;&#x4E0D;&#x662F;&#x6B7B;&#x7684;&#x866B;&#x5375;&#xFF0C;&#x53D1;&#x73B0;&#x5728;&#x539F;&#x6599;&#x5E9F;&#x7269;&#x6216;&#x8868;&#x9762;&#x6C34;&#x63BA;&#x9690;&#x5B62;&#x5B50;&#x866B;&#x5375;&#x56CA;&#x7684;&#x6837;&#x672C;&#x3002;&#x6B64;&#x6982;&#x5FF5;&#x9A8C;&#x8BC1;&#x7814;&#x7A76;&#x662F;&#x7B2C;&#x4E00;&#x6B21;&#x8BC1;&#x660E; PMA &#x7528;&#x4E8E;&#x9884;&#x805A;&#x5408;&#x9176;&#x94FE;&#x53CD;&#x5E94;&#x6CBB;&#x7597;&#x9690;&#x5B62;&#x5B50;&#x866B;&#x5375;&#x56CA;&#x3002;CryptoPMA pcr &#x662F;&#x6709;&#x5438;&#x5F15;&#x529B;&#x7684;&#x529E;&#x6CD5;&#xFF0C;&#x6765;&#x5177;&#x4F53;&#x68C0;&#x6D4B;&#x548C;&#x57FA;&#x56E0;&#x578B;&#x53EF;&#x884C;&#x9690;&#x5B62;&#x5B50;&#x866B;&#x5375;&#x56CA;&#x4E2D;&#x7684;&#x6C34;&#xFF0C;&#x8FD9;&#x662F;&#x5F88;&#x5173;&#x952E;&#x7684;&#x4EBA;&#x4F53;&#x5065;&#x5EB7;&#x98CE;&#x9669;&#x8BC4;&#x4F30;&#x3002;

&#x9690;&#x5B62;&#x5B50;&#x866B;&#x4E19;&#x952D; monoazide PCR&#xFF0C;&#x57FA;&#x56E0;&#x5206;&#x578B;&#x7684;&#x53EF;&#x884C;&#x9690;&#x5B62;&#x5B50;&#x866B;&#x5375;&#x56CA;&#x7684;&#x4E00;&#x79CD;&#x57FA;&#x4E8E;&#x5206;&#x5B50;&#x751F;&#x7269;&#x5B66;&#x7684;&#x6280;&#x672F;&#x3002;

Cryptosporidium is an important waterborne protozoan parasite that can cause severe diarrhea and death in the immunocompromised. The current methods used to monitor for Cryptosporidium oocysts in water are the microscopy-based USEPA methods 1622 and 1623. These methods assess total levels of oocysts in source waters, but do not determine oocyst viability or genotype. Recently, propidium monoazide (PMA) has been used in conjunction with molecular diagnostic tools to identify species and assess the viability of bacteria. The goal of this study was the development of a Cryptosporidium PMA-PCR (CryptoPMA-PCR) assay that includes PMA treatment prior to PCR analysis in order to prevent the amplification of DNA from dead oocysts. The results demonstrated that PMA penetrates only dead oocysts and blocks amplification of their DNA. The CryptoPMA-PCR assay can also specifically detect live oocysts within a mixed population of live and dead oocysts. More importantly, live oocysts, not dead oocysts, were detected in raw waste or surface water samples spiked with Cryptosporidium oocysts. This proof-of-concept study is the first to demonstrate the use of PMA for pre-PCR treatment of Cryptosporidium oocysts. The CryptoPMA-PCR assay is an attractive approach to specifically detect and genotype viable Cryptosporidium oocysts in the water, which is critical for human health risk assessment.

Cryptosporidium propidium monoazide-PCR, a molecular biology-based technique for genotyping of viable Cryptosporidium oocysts.

Cryptosporidium est un parasite protozoaire importante voie d&#x26;#39;eau qui peuvent causer une diarrh&#xE9;e grave et la mort chez les sujets immunod&#xE9;prim&#xE9;s. Les m&#xE9;thodes actuelles utilis&#xE9;es pour surveiller les oocystes de Cryptosporidium dans l&#x26;#39;eau sont les m&#xE9;thodes bas&#xE9;es sur la microscopie EPA 1622 et 1623. Ces m&#xE9;thodes d&#x26;#39;&#xE9;valuer les niveaux totaux d&#x26;#39;oocystes dans les eaux de source, mais ne d&#xE9;terminent pas la viabilit&#xE9; des oocystes ou g&#xE9;notype. R&#xE9;cemment, propidium monoazoture (PMA) a &#xE9;t&#xE9; utilis&#xE9; en conjonction avec des outils de diagnostic mol&#xE9;culaire pour identifier les esp&#xE8;ces et d&#x26;#39;&#xE9;valuer la viabilit&#xE9; des bact&#xE9;ries. L&#x26;#39;objectif de cette &#xE9;tude &#xE9;tait le d&#xE9;veloppement d&#x26;#39;un Cryptosporidium PMA-PCR (CryptoPMA-PCR) qui comprend le traitement PMA avant l&#x26;#39;analyse par PCR, afin d&#x26;#39;&#xE9;viter l&#x26;#39;amplification de l&#x26;#39;ADN &#xE0; partir des oocystes morts. Les r&#xE9;sultats ont d&#xE9;montr&#xE9; que la PMA ne p&#xE9;n&#xE8;tre que les oocystes de morts et d&#x26;#39;amplification des blocs de leur ADN. Le dosage CryptoPMA-PCR peut &#xE9;galement d&#xE9;tecter sp&#xE9;cifiquement oocystes vivants au sein d&#x26;#39;une population mixte d&#x26;#39;oocystes vivants et morts. Plus important encore, les oocystes vivants, des oocystes pas morts, ont &#xE9;t&#xE9; d&#xE9;tect&#xE9;s dans des d&#xE9;chets crus ou les &#xE9;chantillons d&#x26;#39;eau de surface enrichis avec des oocystes de Cryptosporidium. Cette &#xE9;tude de preuve de concept est la premi&#xE8;re &#xE0; d&#xE9;montrer l&#x26;#39;utilisation des PMA pour la pr&#xE9;-PCR de traitement des oocystes de Cryptosporidium. Le dosage CryptoPMA-PCR est une approche int&#xE9;ressante pour d&#xE9;tecter sp&#xE9;cifiquement et les oocystes de Cryptosporidium g&#xE9;notype viables dans l&#x26;#39;eau, ce qui est essentiel pour l&#x26;#39;&#xE9;valuation des risques pour la sant&#xE9; humaine.

Cryptosporidium propidium monoazoture-PCR, une biologie mol&#xE9;culaire bas&#xE9;e sur la technique pour le g&#xE9;notypage des oocystes de Cryptosporidium viables

Cryptosporidium ist eine wichtige Wasserwege-Protozoen-Parasit, der heftigen Durchfall und Tod in der immungeschw&auml;chten verursachen kann. Die aktuellen Methoden zur &Uuml;berwachung der Cryptosporidium-Oozysten im Wasser sind die Mikroskopie-basierte USEPA-Methoden 1622 und 1623. Diese Methoden insgesamt Ebenen von Oozysten im Quellcode Gew&auml;sser zu beurteilen, aber bestimmen nicht Oozyste Lebensf&auml;higkeit oder Genotypus. Vor kurzem wurde Propidium Monoazide (PMA) verwendet in Verbindung mit Molekulare Diagnose-Tools Arten zu identifizieren und bewerten die Lebensf&auml;higkeit der Bakterien. Das Ziel dieser Studie war die Entwicklung einer Cryptosporidium PMA-PCR (CryptoPMA-PCR)-Assay, der PMA-Behandlung vor der PCR-Analyse umfasst, um die Verst&auml;rkung von DNA von Toten Oozysten verhindern. Die Ergebnisse zeigten, dass PMA nur Tote Oozysten und Bl&ouml;cke Verst&auml;rkung von DNA dringt. Der CryptoPMA-PCR-Assay kann auch speziell live Oozysten innerhalb einer gemischten Population von lebenden und Toten-Oozysten erkennen. Noch wichtiger ist, Leben Oozysten, nicht tot Oozysten, gefunden in roher Verschwendung oder Oberfl&auml;che Wasser Proben gespickt mit Cryptosporidium-Oozysten. Diese Proof-of-Concept-Studie ist die erste, die Verwendung von PMA f&uuml;r Pre-PCR Behandlung von Cryptosporidium-Oozysten demonstrieren. Der CryptoPMA-PCR-Assay ist ein attraktives Konzept, speziell zu erkennen und Genotyp lebensf&auml;higen Cryptosporidium-Oozysten im Wasser, das f&uuml;r menschliche Gesundheit-Risikobewertung von entscheidender Bedeutung ist.

Cryptosporidium Propidium Monoazide-PCR, ein Molekularbiologie-basierte Verfahren zur Genotypisierung von lebensf&auml;higen Cryptosporidium-Oozysten.

Cryptosporidium &egrave; un importante a base acquosa protozoo parassita che pu&ograve; provocare diarrea grave e morte nei pazienti immunocompromessi. Gli attuali metodi utilizzati per monitorare le oocisti di Cryptosporidium in acqua sono i metodi USEPA basati su microscopia 1622 e 1623. Questi metodi valutare livelli totali di oocisti nelle acque di fonte, ma non determinano vitalit&agrave; oocisti o genotipo. Recentemente, propidium monoazide (PMA) &egrave; stato usato in combinazione con strumenti di diagnostica molecolari per identificare la specie e valutare la vitalit&agrave; dei batteri. L&#39;obiettivo di questo studio &egrave; stato lo sviluppo di un dosaggio di Cryptosporidium PMA-PCR (CryptoPMA-PCR) che include il trattamento di PMA prima analisi PCR al fine di evitare l&#39;amplificazione del DNA da oocisti morte. I risultati hanno dimostrato che la PMA penetra solo morto oocisti e blocchi di amplificazione del loro DNA. Il dosaggio di CryptoPMA-PCR &egrave; in grado di rilevare anche specificamente oocisti live all&#39;interno di una popolazione mista di oocisti vivi e morte. Ancora pi&ugrave; importante, dal vivo oocisti, oocisti non morte, sono state rilevate in rifiuti crudo o superficie acqua campioni diluiti con le oocisti di Cryptosporidium. Questo studio di proof-of-concept &egrave; il primo a dimostrare l&#39;uso di PMA per il trattamento pre-PCR di oocisti di Cryptosporidium. Il dosaggio di CryptoPMA-PCR &egrave; un approccio interessante per rilevare in modo specifico e oocisti di Cryptosporidium vitali genotipo in acqua, che &egrave; fondamentale per la valutazione dei rischi di salute umana.

Cryptosporidium propidium monoazide-PCR, una tecnica di base di biologia molecolare per la genotipizzazione di oocisti di Cryptosporidium vitali.

&#x30AF;&#x30EA;&#x30D7;&#x30C8;&#x30B9;&#x30DD;&#x30EA;&#x30B8;&#x30A6;&#x30E0;&#x306F;&#x514D;&#x75AB;&#x4E0D;&#x5168;&#x3067;&#x91CD;&#x5EA6;&#x306E;&#x4E0B;&#x75E2;&#x3084;&#x6B7B;&#x3092;&#x5F15;&#x304D;&#x8D77;&#x3053;&#x3059;&#x53EF;&#x80FD;&#x6027;&#x304C;&#x91CD;&#x8981;&#x306A;&#x6C34;&#x7CFB;&#x539F;&#x866B;&#x5BC4;&#x751F;&#x866B;&#x3067;&#x3042;&#x308B;&#x3002;&#x6C34;&#x306E;&#x30AF;&#x30EA;&#x30D7;&#x30C8;&#x30B9;&#x30DD;&#x30EA;&#x30B8;&#x30A6;&#x30E0;&#x30AA;&#x30FC;&#x30B7;&#x30B9;&#x30C8;&#x3092;&#x76E3;&#x8996;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x4F7F;&#x7528;&#x3059;&#x308B;&#x73FE;&#x5728;&#x306E;&#x65B9;&#x6CD5;&#x306F;&#x3001;&#x9855;&#x5FAE;&#x93E1;&#x30D9;&#x30FC;&#x30B9;&#x306E;USEPA&#x30E1;&#x30BD;&#x30C3;&#x30C9;1622&#x3068;1623&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x30E1;&#x30BD;&#x30C3;&#x30C9;&#x306F;&#x3001;&#x30BD;&#x30FC;&#x30B9;&#x6C34;&#x4E2D;&#x306E;&#x30AA;&#x30FC;&#x30B7;&#x30B9;&#x30C8;&#x306E;&#x5408;&#x8A08;&#x30EC;&#x30D9;&#x30EB;&#x3092;&#x8A55;&#x4FA1;&#x3059;&#x308B;&#x304C;&#x3001;&#x30AA;&#x30FC;&#x30B7;&#x30B9;&#x30C8;&#x53EF;&#x80FD;&#x6027;&#x3084;&#x907A;&#x4F1D;&#x5B50;&#x578B;&#x3092;&#x6C7A;&#x5B9A;&#x3059;&#x308B;&#x308F;&#x3051;&#x3067;&#x306F;&#x3042;&#x308A;&#x307E;&#x305B;&#x3093;&#x3002;&#x6700;&#x8FD1;&#x3067;&#x306F;&#x3001;&#x30D7;&#x30ED;&#x30D4;&#x30B8;&#x30A6;&#x30E0;monoazide&#xFF08;PMA&#xFF09;&#x306F;&#x3001;&#x7A2E;&#x3092;&#x8B58;&#x5225;&#x3057;&#x3001;&#x7D30;&#x83CC;&#x306E;&#x751F;&#x5B58;&#x7387;&#x3092;&#x8A55;&#x4FA1;&#x3059;&#x308B;&#x305F;&#x3081;&#x306E;&#x5206;&#x5B50;&#x8A3A;&#x65AD;&#x30C4;&#x30FC;&#x30EB;&#x3068;&#x7D44;&#x307F;&#x5408;&#x308F;&#x305B;&#x3066;&#x4F7F;&#x7528;&#x200B;&#x200B;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x672C;&#x7814;&#x7A76;&#x306E;&#x76EE;&#x7684;&#x306F;&#x3001;&#x6B7B;&#x8005;&#x30AA;&#x30FC;&#x30B7;&#x30B9;&#x30C8;&#x304B;&#x3089;&#x306E;DNA&#x306E;&#x5897;&#x5E45;&#x3092;&#x9632;&#x6B62;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x3001;PCR&#x89E3;&#x6790;&#x306E;&#x524D;&#x306B;PMA&#x51E6;&#x7406;&#x3092;&#x542B;&#x3080;&#x30AF;&#x30EA;&#x30D7;&#x30C8;&#x30B9;&#x30DD;&#x30EA;&#x30B8;&#x30A6;&#x30E0;PMA-PCR&#xFF08;CryptoPMA-PCR&#xFF09;&#x30A2;&#x30C3;&#x30BB;&#x30A4;&#x306E;&#x958B;&#x767A;&#x3067;&#x3057;&#x305F;&#x3002;&#x7D50;&#x679C;&#x306F;&#x3001;PMA&#x3060;&#x3051;&#x6B7B;&#x3093;&#x3060;&#x30AA;&#x30FC;&#x30B7;&#x30B9;&#x30C8;&#x3068;&#x305D;&#x306E;DNA&#x306E;&#x30D6;&#x30ED;&#x30C3;&#x30AF;&#x306E;&#x5897;&#x5E45;&#x3092;&#x8CAB;&#x901A;&#x3059;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x305F;&#x3002; CryptoPMA-PCR&#x30A2;&#x30C3;&#x30BB;&#x30A4;&#x306F;&#x307E;&#x305F;&#x3001;&#x7279;&#x306B;&#x30E9;&#x30A4;&#x30D6;&#x3084;&#x6B7B;&#x3093;&#x3060;&#x30AA;&#x30FC;&#x30B7;&#x30B9;&#x30C8;&#x306E;&#x6DF7;&#x5408;&#x96C6;&#x56E3;&#x5185;&#x3067;&#x306E;&#x30E9;&#x30A4;&#x30D6;&#x30AA;&#x30FC;&#x30B7;&#x30B9;&#x30C8;&#x3092;&#x691C;&#x51FA;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x307E;&#x3059;&#x3002;&#x3055;&#x3089;&#x306B;&#x91CD;&#x8981;&#x306A;&#x306E;&#x306F;&#x3001;&#x30E9;&#x30A4;&#x30D6;&#x30AA;&#x30FC;&#x30B7;&#x30B9;&#x30C8;&#x3001;&#x6B7B;&#x3093;&#x3067;&#x3044;&#x306A;&#x3044;&#x30AA;&#x30FC;&#x30B7;&#x30B9;&#x30C8;&#x306F;&#x3001;&#x751F;&#x30B4;&#x30DF;&#x3084;&#x30AF;&#x30EA;&#x30D7;&#x30C8;&#x30B9;&#x30DD;&#x30EA;&#x30B8;&#x30A6;&#x30E0;&#x30AA;&#x30FC;&#x30B7;&#x30B9;&#x30C8;&#x3067;&#x30B9;&#x30D1;&#x30A4;&#x30AF;&#x306E;&#x5730;&#x8868;&#x6C34;&#x30B5;&#x30F3;&#x30D7;&#x30EB;&#x3067;&#x691C;&#x51FA;&#x3055;&#x308C;&#x305F;&#x3002;&#x3053;&#x306E;&#x6982;&#x5FF5;&#x5B9F;&#x8A3C;&#x7814;&#x7A76;&#x3067;&#x306F;&#x3001;&#x30AF;&#x30EA;&#x30D7;&#x30C8;&#x30B9;&#x30DD;&#x30EA;&#x30B8;&#x30A6;&#x30E0;&#x30AA;&#x30FC;&#x30B7;&#x30B9;&#x30C8;&#x306E;&#x4E8B;&#x524D;-PCR&#x6CBB;&#x7642;&#x306E;&#x305F;&#x3081;&#x306E;PMA&#x306E;&#x4F7F;&#x7528;&#x65B9;&#x6CD5;&#x3092;&#x793A;&#x3059;&#x305F;&#x3081;&#x306B;&#x6700;&#x521D;&#x306E;&#x3082;&#x306E;&#x3067;&#x3059;&#x3002; CryptoPMA-PCR&#x30A2;&#x30C3;&#x30BB;&#x30A4;&#x306F;&#x3001;&#x7279;&#x306B;&#x30D2;&#x30C8;&#x306E;&#x5065;&#x5EB7;&#x30EA;&#x30B9;&#x30AF;&#x8A55;&#x4FA1;&#x306E;&#x305F;&#x3081;&#x306B;&#x91CD;&#x8981;&#x3067;&#x3042;&#x308B;&#x6C34;&#x306B;&#x5B9F;&#x884C;&#x53EF;&#x80FD;&#x306A;&#x30AF;&#x30EA;&#x30D7;&#x30C8;&#x30B9;&#x30DD;&#x30EA;&#x30B8;&#x30A6;&#x30E0;&#x306E;&#x30AA;&#x30FC;&#x30B7;&#x30B9;&#x30C8;&#x3092;&#x691C;&#x51FA;&#x3057;&#x3001;&#x907A;&#x4F1D;&#x5B50;&#x578B;&#x306B;&#x9B45;&#x529B;&#x7684;&#x306A;&#x30A2;&#x30D7;&#x30ED;&#x30FC;&#x30C1;&#x3067;&#x3059;&#x3002;

&#x30AF;&#x30EA;&#x30D7;&#x30C8;&#x30B9;&#x30DD;&#x30EA;&#x30B8;&#x30A6;&#x30E0;&#x30D7;&#x30ED;&#x30D4;&#x30B8;&#x30A6;&#x30E0;Monoazide-PCR&#x3001;&#x751F;&#x304D;&#x305F;&#x30AF;&#x30EA;&#x30D7;&#x30C8;&#x30B9;&#x30DD;&#x30EA;&#x30B8;&#x30A6;&#x30E0;&#x306E;&#x30AA;&#x30FC;&#x30B7;&#x30B9;&#x30C8;&#x306E;&#x30B8;&#x30A7;&#x30CE;&#x30BF;&#x30A4;&#x30D4;&#x30F3;&#x30B0;&#x306E;&#x305F;&#x3081;&#x306E;&#x5206;&#x5B50;&#x751F;&#x7269;&#x5B66;&#x30D9;&#x30FC;&#x30B9;&#x306E;&#x6280;&#x8853;

&#xC6C0;&#xC744; immunocompromised&#xC5D0; &#xC2EC;&#xD55C; &#xC124;&#xC0AC; &#xBC0F; &#xC8FD;&#xC74C;&#xC744; &#xC77C;&#xC73C;&#xD0AC; &#xC218; &#xC788;&#xB294; &#xC911;&#xC694; &#xD55C; waterborne protozoan &#xAE30;&#xC0DD;&#xCDA9; &#xC774;&#xB2E4;. &#xC6C0; oocysts &#xBB3C;&#xC5D0; &#xB300; &#xD55C; &#xBAA8;&#xB2C8;&#xD130;&#xB9C1; &#xD558;&#xB294; &#xB370; &#xC0AC;&#xC6A9; &#xD558;&#xB294; &#xD604;&#xC7AC; &#xBA54;&#xC11C;&#xB4DC;&#xB294; 1622 &#xBC0F; 1623 &#xD604;&#xBBF8;&#xACBD;-&#xAE30;&#xBC18; USEPA &#xBC29;&#xBC95;. &#xC774;&#xB7EC;&#xD55C; &#xBC29;&#xBC95;&#xC740; oocysts &#xC18C;&#xC2A4; &#xBC14;&#xB2E4;&#xC5D0;&#xC11C;&#xC758; &#xC804;&#xCCB4; &#xC218;&#xC900;&#xC744; &#xD3C9;&#xAC00; &#xD558;&#xC9C0;&#xB9CC; oocyst &#xC0DD;&#xC874; &#xB610;&#xB294; &#xC720;&#xC804;&#xC790; &#xD615;&#xC744; &#xACB0;&#xC815; &#xD558;&#xC9C0; &#xC54A;&#xC2B5;&#xB2C8;&#xB2E4;. &#xCD5C;&#xADFC;, propidium monoazide (PMA) &#xC218; &#xC885; &#xC2DD;&#xBCC4; &#xD558; &#xACE0; &#xBC15;&#xD14C;&#xB9AC;&#xC544;&#xC758; &#xC0DD;&#xC874; &#xB2A5;&#xB825; &#xD3C9;&#xAC00; &#xBD84;&#xC790; &#xC9C4;&#xB2E8; &#xB3C4;&#xAD6C;&#xC640; &#xD568;&#xAED8;&#xC5D0;&#xC11C; &#xC0AC;&#xC6A9; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC774; &#xC5F0;&#xAD6C;&#xC758; &#xBAA9;&#xD45C;&#xB294; &#xBC29;&#xC9C0; &#xC8FD;&#xC740; oocysts&#xC5D0;&#xC11C; DNA &#xC99D;&#xD3ED; &#xD558;&#xAE30; &#xC704;&#xD558;&#xC5EC; PCR &#xBD84;&#xC11D; &#xC804;&#xC5D0; PMA &#xCE58;&#xB8CC; &#xD3EC;&#xD568; &#xC6C0;&#xC744; PMA PCR (CryptoPMA-PCR) &#xBD84;&#xC11D; &#xACB0;&#xACFC; &#xAC1C;&#xBC1C; &#xD588;&#xB2E4;. &#xACB0;&#xACFC; PMA&#xC758; DNA&#xB9CC; &#xC8FD;&#xC740; oocysts &#xBC0F; &#xBE14;&#xB85D; &#xC99D;&#xD3ED; &#xCE68;&#xD22C; &#xC2DC;&#xC5F0; &#xD588;&#xB2E4;. CryptoPMA-PCR &#xBD84;&#xC11D; &#xACB0;&#xACFC; &#xB77C;&#xC774;&#xBE0C;&#xC640; &#xC8FD;&#xC740; oocysts&#xC758; &#xD63C;&#xD569; &#xB41C; &#xC778;&#xAD6C; &#xB0B4;&#xC758; &#xB77C;&#xC774;&#xBE0C; oocysts &#xAC10;&#xC9C0; &#xAD6C;&#xCCB4;&#xC801;&#xC73C;&#xB85C; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xB354; &#xC911;&#xC694; &#xD55C; &#xAC83;&#xC740;, &#xB77C;&#xC774;&#xBE0C; oocysts, &#xC544;&#xB2C8; &#xC8FD;&#xC740; oocysts &#xC6D0;&#xC2DC; &#xD3D0;&#xAE30;&#xBB3C; &#xB610;&#xB294; &#xD45C;&#xBA74;&#xC5D0;&#xC11C; &#xBC1C;&#xACAC; &#xB41C; &#xC0D8;&#xD50C; &#xC6C0; oocysts&#xB85C; &#xC544;&#xAD70;&#xC744; &#xBB3C;. &#xC774; &#xAC1C;&#xB150; &#xC99D;&#xBA85; &#xC5F0;&#xAD6C;&#xB294; &#xBA3C;&#xC800; &#xC6C0; oocysts&#xC758; pre-PCR &#xCE58;&#xB8CC;&#xC5D0; &#xB300; &#xD55C; PMA&#xC758; &#xC0AC;&#xC6A9; &#xBC29;&#xBC95;&#xC744; &#xC124;&#xBA85; &#xD558;&#xAE30;. CryptoPMA-PCR &#xBD84;&#xC11D; &#xACB0;&#xACFC; &#xAD6C;&#xCCB4;&#xC801;&#xC73C;&#xB85C; &#xAC10;&#xC9C0; &#xD558;&#xB294; &#xB9E4;&#xB825;&#xC801;&#xC778; &#xC811;&#xADFC; &#xBC0F; &#xC720;&#xC804;&#xC790; &#xD615; &#xAC00;&#xB2A5;&#xD55C; &#xC6C0; oocysts &#xC778;&#xAC04;&#xC758; &#xAC74;&#xAC15; &#xC704;&#xD5D8; &#xD3C9;&#xAC00; &#xB300; &#xD55C; &#xC911;&#xC694; &#xD55C; &#xBB3C;&#xC5D0;&#xC11C;.

&#xC6C0; propidium monoazide-PCR, &#xAC00;&#xB2A5;&#xD55C; &#xC6C0; oocysts &#xC720;&#xC804;&#xD615; &#xBD84;&#xC790; &#xC0DD;&#xBB3C;&#xD559; &#xAE30;&#xBC18; &#xAE30;&#xC220;.

Cryptosporidium es un protozoario par&#xE1;sito importantes transmitidas por el agua que puede causar diarrea severa y la muerte en los pacientes inmunodeprimidos. Los m&#xE9;todos actuales utilizados para detectar ooquistes de Cryptosporidium en el agua son los m&#xE9;todos basados &#x200B;&#x200B;en microscop&#xED;a USEPA 1622 y 1623. Estos m&#xE9;todos de evaluar los niveles totales de ooquistes en las fuentes de agua, pero no determinar la viabilidad de los ooquistes o genotipo. Recientemente, propidio monoazide (PMA) se ha utilizado en conjunci&#xF3;n con moleculares herramientas de diagn&#xF3;stico para identificar las especies y evaluar la viabilidad de las bacterias. El objetivo de este estudio fue el desarrollo de un Cryptosporidium PMA-PCR (CryptoPMA-PCR) que incluye el tratamiento de PMA antes del an&#xE1;lisis de PCR con el fin de evitar la amplificaci&#xF3;n de ADN de ooquistes muertos. Los resultados demostraron que el PMA s&#xF3;lo penetra ooquistes muertos y amplificaci&#xF3;n de los bloques de su ADN. El ensayo CryptoPMA-PCR tambi&#xE9;n se puede detectar ooquistes en concreto en vivo dentro de una poblaci&#xF3;n mixta de oocistos vivos y muertos. M&#xE1;s importante a&#xFA;n, los ooquistes en vivo, los ooquistes no muertos, se han detectado en los residuos de crudos o las muestras de aguas superficiales enriquecidas con ooquistes de Cryptosporidium. Este estudio de prueba de concepto es el primero en demostrar el uso de los PMA para el tratamiento pre-PCR de ooquistes de Cryptosporidium. El ensayo CryptoPMA-PCR es un enfoque atractivo para la detecci&#xF3;n espec&#xED;fica y ooquistes de Cryptosporidium genotipo viables en el agua, lo cual es cr&#xED;tico para la evaluaci&#xF3;n del riesgo para la salud humana.

Cryptosporidium propidio Monoazide-PCR, uno de Biolog&#xED;a Molecular basada en la t&#xE9;cnica de tipificaci&#xF3;n gen&#xE9;tica de las viables ooquistes de Cryptosporidium

Saliva has an important advantage over serum as a medium for antibody detection due to non-invasive sampling, which is critical for community-based epidemiological surveys. The development of a Luminex multiplex immunoassay for measurement of salivary IgG and IgA responses to potentially waterborne pathogens, Helicobacter pylori, Toxoplasma gondii, Cryptosporidium, and four noroviruses, involved selection of antigens and optimization of antigen coupling to Luminex microspheres. Coupling confirmation was conducted using antigen specific antibody or control sera at serial dilutions. Dose-response curves corresponding to different coupling conditions were compared using statistical tests. Control proteins in the specific antibody assay and a separate duplex assay for total immunoglobulins G and A were employed to assess antibody cross-reactivity and variability in saliva composition. 200 saliva samples prospectively collected from 20 adult volunteers and 10 paired sera from a subset of these volunteers were used to test this method. For chronic infections, H. pylori and T. gondii, individuals who tested IgG seropositive using commercial diagnostic ELISA also had the strongest salivary antibody responses in salivary antibody tests. A steep increase in anti-norovirus salivary antibody response (immunoconversion) was observed after an episode of acute diarrhea and vomiting in a volunteer. The Luminex assay also detected seroconversions to Cryptosporidium using control sera from infected children. Ongoing efforts involve further verification of salivary antibody tests and their application in larger pilot community studies.

Development of a multiplex microsphere immunoassay for the quantitation of salivary antibody responses to selected waterborne pathogens.

Phospholipids and lung surfactant proteins are known to be recycled within the lung alveolus mainly by uptake into type II epithelial cells that secrete lipid-enriched lung surfactant. Dipalmitoyl phosphatidylcholine (DPPC) is the major component of lung surfactant lipids and cholesterol is the second most abundant. However, cholesterol turnover in vivo has not been measured and it is not known how long steroidal compounds persist in the lung in intact animals. Here we report on experiments in which radiolabeled cholesterol was instilled into the lungs of rats, then at various postinstillation periods, radioactive sterols in lavage fluid, and in postlavage whole lungs were measured in individual animals. Radioactive sterols in the lungs remained high for a week and were still detectable 46 days later. The clearance rate during the initial postinstillation week was approximately 10% per day. Both radioactive free and esterified sterols were recovered from bronchoalveolar lavage fluid and postlavage lungs.

Clearance in vivo of instilled [h]cholesterol from the rat lung.

The U.S. EPA developed a sample concentration and preparation assay in conjunction with the total culturable virus assay for concentrating and measuring culturable viruses in source and drinking waters as part of the Information Collection Rule (ICR) promulgated in 1996. In an effort to improve upon this method, the U.S. EPA recently developed Method 1615: Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Method 1615 uses a culturable virus assay with reduced equipment and labor costs compared to the costs associated with the ICR virus method and introduces a new molecular assay for the detection of enteroviruses and noroviruses by reverse transcription-quantitative PCR. In this study, we describe the optimization of several new components of the molecular assay and examine virus recovery from ground, reagent-grade, and surface water samples seeded with poliovirus type 3 and murine norovirus. For the culturable virus and molecular assays, mean poliovirus recovery using the complete method was 58% and 20% in groundwater samples, 122% and 39% using low-titer spikes in reagent-grade water, 42% and 48% using high-titer spikes in reagent-grade water, and 11% and 10% in surface water with high turbidity, respectively. Murine norovirus recovery by the molecular assay was 30% in groundwater samples, less than 8% in both low- and high-titer spikes in reagent-grade water, and 6% in surface water with high turbidity. This study demonstrates the effectiveness of Method 1615 for use with groundwater samples and highlights the need for further research into its effectiveness with surface water.

Development and evaluation of EPA method 1615 for detection of enterovirus and norovirus in water.

The objective of this study was to compare three nucleic acid extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) approaches for norovirus (NoV) detection in drinking water with respect to performance, costs, and analysis time. The approaches evaluated were: (A) an approach that utilizes the QIAamp DNA Blood Mini Kit and multiplex primers and probes for detection; (B) a procedure which includes the NucliSENS Magnetic Extraction Kit and other components of a proposed European Union standard method for NoV detection in foods; and (C) a commercialized assay which uses NucliSENS extraction and Cepheid SmartCycler® technologies. Each approach was evaluated by most probable number (MPN) analysis for detection of GI.1 and GII.4 NoVs from human stool. Furthermore, recoveries of spiked primary effluent in tap water concentrates were compared for each approach. Few significant differences were observed between approaches with regard to performance. However, Approach C was the most time consuming and expensive to perform. This research presents a case study of how molecular-based approaches for detection of NoVs can be compared and how various factors may play a role in which approach laboratories choose to employ.

Comparison of nucleic acid extraction and reverse transcription-qPCR approaches for detection of GI and GII noroviruses in drinking water.

Norovirus infection is the most common cause of acute gastroenteritis in developed countries. Developing an assay based on a non-invasive biomarker for detecting incident norovirus infections could improve disease surveillance and epidemiological investigations. This project involved analysis of IgA and IgG norovirus-specific antibody responses in saliva samples from a Norwalk virus (Genogroup I, genotype 1 norovirus) challenge study involving infected and symptomatic, and non-infected asymptomatic individuals. Saliva was collected at the challenge, and two weeks and 40 days post-challenge. Samples were analyzed using the Luminex fluorometric and Meso Scale Discovery (MSD) electrochemiluminescence immunoassays. Recombinant P domains of Norwalk virus capsid protein, as well as similar recombinant proteins of two genogroup II noroviruses (VA387 and VA207) were used as antigens. Immunoconversions were defined as >4-fold increase in antibody responses to the norovirus antigens. Various sample pre-treatment options, buffers, saliva dilution ratios, and data adjustment approaches to control for sample-to-sample variability in saliva composition were compared using the Luminex assay. The results suggest that adjusting responses to the norovirus antigens for responses to the protein purification tag, glutathione-S-transferase (GST), significantly improved the odds of producing a correct immunoconversion test result. IgG-based tests were more accurate compared to IgA-based tests. At optimal conditions, both Luminex and MSD assays for Norwalk-specific IgG antibodies correctly identified all infected and non-infected individuals. There was no evidence of cross-reactivity of anti-Norwalk virus antibodies with genogroup II noroviruses. These results suggest that salivary antibody responses can be used for the detection of incident infections with Norwalk virus in prospective surveys.

Application of salivary antibody immunoassays for the detection of incident infections with Norwalk virus in a group of volunteers.

There are numerous pathogens that can be transmitted through water. Identifying and understanding the routes and magnitude of exposure or infection to these microbial contaminants are critical to assessing and mitigating risk. Conventional approaches of studying immunological responses to exposure or infection such as Enzyme-Linked Immunosorbent Assays (ELISAs) and other monoplex antibody-based immunoassays can be very costly, laborious, and consume large quantities of patient sample. A major limitation of these approaches is that they can only be used to measure one analyte at a time. Multiplex immunoassays provide the ability to study multiple pathogens simultaneously in microliter volumes of samples. However, there are several challenges that must be addressed when developing these multiplex immunoassays such as selection of specific antigens and antibodies, cross-reactivity, calibration, protein-reagent interferences, and the need for rigorous optimization of protein concentrations. In this study, a Design of Experiments (DOE) approach was used to optimize reagent concentrations for coupling selected antigens to Luminex™ xMAP microspheres for use in an indirect capture, multiplex immunoassay to detect human exposure or infection from pathogens that are potentially transmitted through water. Results from Helicobacter pylori, Campylobacter jejuni, Escherichia coli O157:H7, and Salmonella typhimurium singleplexes were used to determine the mean concentrations that would be applied to the multiplex assay. Cut-offs to differentiate between exposed and non-exposed individuals were determined using finite mixed modeling (FMM). The statistical approaches developed facilitated the detection of Immunoglobulin G (IgG) antibodies to H. pylori, C. jejuni, Toxoplasma gondii, hepatitis A virus, rotavirus and noroviruses (VA387 and Norwalk strains) in fifty-four diagnostically characterized plasma samples. Of the characterized samples, the detection rate was 87.5% for H. pylori, and 100% for T. gondii assays and 89% for HAV. Further, the optimized multiplex assay revealed exposure/infection to several other environmental pathogens previously uncharacterized in the samples.

Shannon M. Griffin

National Exposure Research Laboratory

U.S. Environmental Protection Agency

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens

Shannon M. Griffin

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}National Exposure Research Laboratory

U.S. Environmental Protection Agency

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens

National Exposure Research Laboratory